In one of the movie's early scenes, set in the animal rescue center where Penny adopts Bolt, a poster can be seen with the year 2008 written on it.
Yet, in the next scene, which according to the film is taking place five years later, Bolt can be seen reading a magazine dated March 2008. That would be the same year Bolt was adopted. This is an obvious impossibility.
The front of the train is visible going under the bridge just before Bolt, Rhino, and Mittens jump. Later, both ends of the train are visible. The train is much shorter than the length of the time between the first sighting of it and them jumping would suggest.
(at around 6 mins) On the "Bolt" TV series episode at the beginning of the movie, as Bolt is holding the car containing the guard from the bridge, we see the approaching helicopters, then we see the bridge as seen from the approaching helicopters. From that perspective, there is no car hanging from the bridge.
Near the end of the movie, Penny is getting ready for the TV show episode. You can clearly see that the cables behind her are taut. In the very next shot, the cables have slack in them and they are being pulled taut.
The scene in which Mittens tries to get Bolt to stay in Las Vegas to live takes place in a desert-type field, not far from some alleyways - presumably south of Mandalay Bay based on the background shots. While it's true there are some dirt lots south of Mandalay Bay, there are no alleyways close to them. Likewise, this desert-type area seems to stretch on endlessly, but there are no empty lots that stretch on this far in the area - as the area being occupied is (in real life) surrounded by roads and hotel/casinos on every end.
Bolt calls Dr. Calico "The Green-Eyed Man". Even though a great percentage of people think the opposite, dogs are not actually colorblind. However, they do see colors differently than how humans do. Dogs don't seem to be able to see reds or greens very well, so their world probably exists in various shades of yellow and blue. Because of this, there is no way that he could recognize Calico's eye as green. However, he would have heard Penny calling him "The Green-Eyed Man" a lot and is likely to have repeated this. Yet, Bolt is able to recognize blood as "this red liquid" when he injures himself.
The CSX diesel locomotive pulling the train that Bolt swings onto from the bridge is an EMD SD70M, #432. CSX did not number any of their SD70Ms in the 400s. CSX numbered their SD70Ms from 4675 to 4699. CSX locomotive #432 is, in reality, a CW44AC (CSX's designation for an AC4400CW).
In the beginning scene, where Penny and Bolt are acting out the chase scene on the playback monitor, the producers are enraged over a boom mic showing up in the shot. Yet seconds earlier during the actual scene, you don't see the boom mic in the upper right corner. This is not a goof, as the playback monitor may show areas near the edges of the picture that would be cropped in the actual broadcast.
(at around 20 mins) In the "Cliffhanger" scene, Penny rolls a penny to distract the guards. The closeup of the back of the penny is missing Abraham Lincoln sitting in his chair. This happens frequently in cartoons. Since there are laws against depicting currency too realistically in reference books etc., the animators are being overly cautious to avoid accusations of counterfeiting.
(at around 1h) At a shelter, a tank of helium explodes. While helium itself is not flammable, the helium tank is filled with a compressed gas. When the valve gets knocked off the helium tank, the compressed gas rapidly escapes and turns the tank into a missile. The tank flies uncontrolled out of the building, and hits a sign post, knocking it down. The sign post falls onto a truck carrying a larger tank (presumably a fuel tank), which can be seen correctly labeled with a flammable liquid placard.
(at around 10 mins) The director is upset because a boom microphone is visible in the frame and "the dog could have seen that." Regardless of what the camera captured, the microphone and its operator were out in the open on the set itself, where Bolt could have seen them during filming, even if they were edited out later.
(at around 1h) After escaping from the shelter, the 3 animals chase a vehicle and then Bolt grabs Rhino's ball with his mouth and jumps into the vehicle. This should not be possible because in any other scene, it is obvious that the ball is too big for Bolt to hold in his jaws. In fact at no other time do we see Bolt hold the ball in his mouth.
Near the beginning of the film, Calico is sitting with a cat on his right shoulder. Their reflection is shown in the glasses of a man directly opposite. The reflection shows Calico and the cat in the same position instead of being reversed, as a reflection would be.
(at around 38 mins) In the Uhaul truck, just before Mittens is about to hit Bolt with a baseball bat that she found in the truck, she falls backwards, dislodging a box. This box falls on Bolt and is filled with Styrofoam, and apparently nothing else.
After Bolt had fallen into the cardboard box it is carried away with a smaller box and placed into a truck together with other boxes. When comparing the bruises and cracked edges of the boxes one realizes that the boxes are only resized copies of each other.
When the man hears Bolt's barking coming from a shipping box, he takes a knife and slices the tape that goes across the top of the box just once. However, in the very next shot, the tape is clearly shown as having been cut along the sides as well.
(at around 50 mins) When Mittens is on the tree branch and is still tied with the leash to Bolt on the ground, she walks forward and loops the leash onto a broken branch. The leash runs down behind the main branch on her left side, there is a smaller branch ahead of her sprouting out on her left (we see this in an overhead shot.) When she moves forward to loop the leash on the broken branch, the leash somehow moves through this branch on her left instead of getting stuck behind it.
In the beginning of the movie during the chase scene, when Bolt places the time bomb on the chasing henchmen's helmet, Penny's image is reflected on his visor. After he takes the time bomb down, Penny's reflection on his helmet visor is gone.
The goof items below may give away important plot points.
(at around 1h 28 mins) When Penny takes a Polaroid picture of herself with Bolt, Rhino, and Mittens at the end of the movie, Mittens was sitting on the couch with her head facing Penny's mother and with her rear end facing Rhino before, during, and after the picture. When the developed picture is displayed after Penny leaves the house to play with Bolt outside, Mittens' head is now next to Rhino in the picture.
(at around 1h 26 mins) When Penny is loaded into the ambulance, the cot is placed towards the passenger side of the ambulance. The cot retention mechanism in an ambulance is on the driver's side. In a following shot through the open doors of the ambulance, it also shows the cot in a raised position. Cots are collapsed when placed in an ambulance.