(at around 26 mins) After watching TV, when the puppies are going off to bed, when Pongo counts puppies 8 through 14 (seven puppies), there are only five puppies, including Rolly, who actually go through the door.
(at around 15 mins) After Cruella de Vil leaves the house after looking for the puppies that haven't been born yet, Roger comes from the attic singing the Cruella de Vil theme, holding a long cigarette. When he reaches the bottom of the stairs, the cigarette vanishes.
(at around 45 mins) When Jasper reaches for the bottle to throw at the escaping cat it still has wine in it, but when the bottle breaks against the wall, the bottle is empty and no wine spills out when the bottle shatters.
(at around 6 mins) When Pongo opens the clock to change the time to 5:18, for his evening constitutional, he starts to bark, letting Roger know it's time for his walk. But he forgot to close the clock door. But when Roger looks up, it's mysteriously closed.
(at around 1h 14 mins) When Cruella is chasing the van, she is knocked down the hill next to the bridge. The shot of her crashing through the fence shows that the van has disappeared. It then reappears when it crosses the bridge.
When Sgt. Tibs first enters Hell Hall, we see the main hall. The stairway is to the left, it goes up to a landing, then turns right. When the Baduns chase Tibs and the puppies, the hall has been mirrored - the stairs are on the opposite side of the hallway, and they turn left.
(at around 35 mins) After the art lady in the attic yells "Prissy, come in here!", she pulls Prissy inside and the window closes. In the next shot when Prissy knocks over the painting as she runs over to the other side of the attic, the window she was at is opened again.
Patch's right ear is solid black, and his left ear spotted. However, sometimes the left ear is solid white, and sometimes it's solid black. In the same scene where Patch turns into Lucky, his ear goes from spotted to white, and in the following scene in the Colonel's barn, both of his ears are solid black.
(at around 22 mins) When Perdita and Pongo are nuzzling after the news that Cruella is not going to get any of their puppies, before it changes to the scene to reveal the puppies nursing, the tags on Perdita and Pongo's collars vanish.
(at around 31 mins) Cruella's phone when it was first picked up (to be Jasper), has a face of a devil with its eyes open and wide-mouth smiling. Yet when she hangs up and calls Anita, the face has its eyes closed and is heavily frowning.
(at around 4 mins) In the beginning, Roger is shown to wear a wrist-watch which disappears when he first lights his pipe, and reappears in the next scene. When he goes to the park assuming it was hidden under his coat sleeve, it is gone when he takes off his coat to give it to a wet Anita.
(at around 49 mins) When Cruella takes the bottle of alcohol away from Jasper's mouth to throw it in the fire, she grabs it upside down, but when she's about to throw it, she is holding it right-side up.
(at around 10 mins) When Pongo pulls Roger and Anita into the pond Perdita remains on land. Yet when Roger is helping Anita out, Perdita walks out of the pond and Pongo, next to her is shown to be perfectly dry.
(at around 59 mins) The Captain rears up his left hind leg to kick the Baduns, but from the back, one sees him kick with his right leg. He then kicks Jasper first, who had been lined up behind his left leg.
(at around 36 mins) During the Twilight Bark, there is a scene showing St Paul's Cathedral across the other side of the River Thames. This means that the view must be facing north. However there is a full Moon near the horizon in the background, which would not be seen in a north-facing view.
Pongo and Perdita set off from London to Suffolk (north), but when dawn breaks we see them running left to right across the screen, with the dawn breaking in the background. Since dawn is in the southeast in England in the winter, this means they are running roughly southwest.
(at around 1h 3 mins) While the puppies and trudging through the blizzard, they are heading into the wind. Then the Collie finds them, and they turn around to go back the other way and are still heading into the wind.
(at around 34 mins) When Pongo starts howling to send out the last part of the Twilight Bark, his four paws temporarily do not make contact with the ground; giving the image of walking on thin air before being pulled back to the ground by his owner.
In the title screen, you can see that all of the pictures are different poses of Pongo. But looking closer, some are clearly designed to be Perdita, especially when in the same pose together, when she was designed/spotted to be Pongo; despite the slimmer body.
The goof items below may give away important plot points.
(at around 1h 14 mins) During final car chase, Jasper and Horace see the moving truck approaching the intersection from their right, but when the vehicles collide, the moving truck has come from their left.