When Lightning first goes to Radiator Springs and tears up the street, he is caught in the middle of the road on a electric wire between 2 poles. When that area is shown again there is no wire going across the road at any point.
When Lightning McQueen is sentenced to pave the road as punishment, Sally says that he "has the horsepower" when in fact towing a trailer has much more to do with Torque than Horsepower, making a race car like McQueen unsuited to towing such a large object as Bessy.
When Lightning McQueen wakes up in the impound yard, the boot is on the driver's side front tire. Mater hooks up to Lightning's rear to tow him in. He should have hooked to the front since one of front tires was incapacitated.
When McQueen, Chick, and The King cross the finish line during the first race, McQueen can be seen, on close inspection, sticking out his tongue out and downward, curling it under considerably. When the race officials review the instant replay, his tongue is shown sticking nearly straight out and level.
After Lightning catches up with a truck he thought was Mack (to discover it's a Peterbilt), he turns left on the T-junction from what is clearly an asphalt road (the one he just came from). During the actual turn, however, this road suddenly seems to be made of gravel or dirt.
After Mater tips two tractors, McQueen attempts to follow suit. When the camera pulls back to show all of the tractors tipping, the two Mater tipped initially are nowhere to be seen. Mater has not moved appreciably, so the tractors he tipped should have been on-screen right next to him.
As Mini and Van are approaching Radiator Springs, there is nothing on the sides of the road where the damage begins. As they continue through town everyone is in front of them or following them as they pass. After they leave there is a zoom shot down the damaged road. There are now 2 "END CONSTRUCTION ZONE" signs, one on either side of the road. When McQueen "finishes the road" moments later, the signs are gone. The signs stay gone in every available shot except one. When McQueen does finish the road and the townsfolk think he left the signs have suddenly returned.
When McQueen arrives at the tie-breaker race, the announcer comments on the fact that McQueen is wearing white-wall tires. Yet in a couple of shots immediately before, he is clearly NOT wearing white-walls.
At the end of the first race Lightning blows both of his back tires and is riding on his rims, but when he is getting his tires changed out during his interview the tires being changed out are back on the rims and there is no evidence of blown tires.
During the end of the final race, when King crashes and Lighting is giving him the rear bump, we can see that the front and rear tires of King are flat, but when they arrive at the Dinoco camp, King's tires appear perfectly fine.
After Lightning McQueen learns about Doc Hudson's past, the shot goes to Flo's V8 Cafe with the road in front of it having its old faded look, but in the next shot the road having the new tar that McQueen paved.
At the beginning of the films while the commentators are speaking there are long, colored arrows that separately point to Chick, Weathers, and McQueen. Weathers' and McQueen's arrows point downward center, but Chick's arrow points behind him.
In the second race, when Chick makes Lightning lose control, McQueen passes him in reverse. So McQueen's race position is now second. But a few moments later, we see the board at lap 190 and we see number 43 in the lead and the top part of number 86 (Chick's number) as position two. However, the position board is not updated until cars pass the start/finish line.
Contrails in the movie look like tire marks, even though the movie features a bi-plane and fighter jets, which shouldn't leave tire mark contrails. This is most probably an intentional joke, rather than a goof (see trivia). (Jokes notwithstanding, a contrail can theoretically resemble a tire mark, if disrupted by air currents in just the right way.)
After Lightning chases down a truck and discovers it is not Mack, he wants to get back to the Interstate. At a first glance it may seem as if he turns and goes back the way he came (to the Interstate), but arrives to Radiator Springs. However, a closer look reveals that he makes a U-turn after turning right (to watch the truck leave), which means that eventually he went left on the T-junction (possibly becoming confused in the dark), instead of going back.
It is announced at the beginning of the Dinoco 400 that Chick Hicks has never managed to beat the King, even though he ties in points with him and McQueen in the season. Apparently, the announcers meant that Chick never won the Cup (which is true), and not that he never won an individual race. In fact, the announcers' exact words were: "And as always in the second place spot we find Chick Hicks." This can be interpreted either way.
Lightning has a full re-spray just before he leaves Radiator Springs, including white sections with a yellow lightning bolt. But when we see him in the race, he's back in his old colors, and when he gets back to Radiator Springs he's in his new colors again. Evidently, cars change their paint job as readily as humans change clothes, which Ramone demonstrates repeatedly throughout the movie. McQueen's changes are consistent with the plot and the character - he likes his new paint job, but can't use it in a race, because it doesn't feature his sponsor's ads, in particular.
As the mini-van with the mattress passes Mack on the interstate, it has its left turn signal on, when in fact it goes right, toward the exit ramp. This appears to be intentional. The mini-van appears to have an implied "dopey" character - adding to the novelty that the little mini-van has somehow bested Mac in traveling.
The entire premise of the film revolves around the fact that Lightning, Hicks, and the King are tied for the top spot in the points in the closing laps of the final race. It's been argued that according to the rules of the much similar Nextel Cup Series, the chances for such a tie would be astronomically small. Evidently, the rules of the fictional Piston Cup are different enough to make such a situation much more likely, even if not common (the announcer did say that this was the first three-way tie in Cup's history).
After the first race, McQueen is being interviewed by a reporter as his pit crew changes his tires from racing slicks to wide street tires with treads. They change the left side, but quit while doing the right side, however the next shot has treads on the right side. In fact, "Chuck" has just finished loosening the lugnuts on the front right when he throws done his impact wrench and quits.
When Lightning is loaded onto Mack after the first race, they are using video to communicate back and forth. Lightning is trying to convince Mac to drive all night to get to California. Inside the trailer, Lightning's monitor shows all of Mack's face, yet when the camera view switches outside the trailer, there is no camera for Mac to have been looking into.
The goof items below may give away important plot points.
Lightning McQueen is being identified as the first rookie to ever win the Piston Cup, however, when Doc Hudson's trophies were discovered they were engraved "1951, 1952, 1953". Doc's license plate identifies him as a 1951. That would have made him a rookie when he won his 1951 Piston Cup.
At the start of the film, The King has brown eyes. However, when he opens them after crashing, they have somehow turned blue. During the credits when he is seen in the museum with his wife, they have turned brown again.
In the final tie-breaker race, when Lightning stops just before the finish line, he is about a quarter of his body length from it. But in the subsequent wide shot he is almost adjacent to it, and in the next shot when he's about to move backwards, he's almost half of his body length away from it.
During the tie-breaker race, Lightning spots the Radiator Springs residents in the crew pits. In the immediately following closer shot there are several noticeable differences: Mack no longer wears a headset, Ramone stands behind Flo and Mater (he was just standing between them), Sarge and Fillmore move closer to the others, and Sheriff shifts closer to Doc. In the next shot, showing the same group from behind, the positions of the cars change again slightly and Mack's headset is back.
Before the final race, when Chick flashes the silver C at Lightning, the reflected C is open toward the rear of Lightning but it should be open to the front, just like it is on Chick. This backwards reflection is also shown during the race when Chick uses it to blind Lightning temporarily.
(at around 1h 40 mins) Chick disappears from the scene in which McQueen's brakes stop. He should be in the background, behind McQueen. At that moment, McQueen is braking, the King can be seen in the right corner, on grass, but Chick has disappeared.
During the second race, when Lightning slams on his brakes to prevent himself from crossing the finish line, a female voice is heard saying "What's he up to, Doc?". It has been claimed that no character could've said those words, but the voice clearly belongs to Flo, who is standing on the Doc's left side (as seen in the previous shot).