Continuity mistake: It was briefly stated in the second film that the T-Virus was created by Dr. Ashford to cure his daughter Angela and that the Red and White Queens were based on her. This movie contradicts that by suggesting that Dr. Marcus created the T-Virus to cure his daughter Alicia, and that the Red/White Queens were modeled after her. While the rest of the movie stays true to the continuity of the previous films, this rather obvious ret-con is still a major continuity gaff.
Continuity mistake: At the first overpass Alice tries to use the motorcycle that's just sitting there, strangely clean. She doesn't have her prints in their system so the bike won't start and she gets zapped. She gets captured by Dr Isaacs and cuts off his left hand to escape on a motorcycle carried on his armored vehicle. Later at another destroyed flyover, she gets back onto the bike and rides towards Raccoon City. No fingerprint or hand required.
Other mistake: Alice is running behind the armored cars. Once she manages to get on top of the car, suddenly the zombies that were only behind her and nowhere near the car are found at the sides too, attacking a guard and later Alice as well. Not possible considering if zombies were able to get there, they would have killed Alice while she was running.
Continuity mistake: There is a scene of Alice being dragged by a line behind Isaacs' tank ahead of a horde of zombies. There is no other line next to running Alice. When she climbs upon the tank to attack the guard, another line with torn hands is seen being dragged on the road, where Alice was dragged.