Plot hole: When L.J. Is bitten he manages to suppress becoming a zombie for 1-2 days. When Carlos is bitten by L.J. he is infected so quickly and talks about it "being too late for him" (whereas in the previous film he had 3 hours before getting the antidote). Why is this different now and why is Carlos not still carrying the antidote in him?
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Continuity mistake: After the Zombies are unleashed in the desert scene, we see Carlos crawling under the truck. At first his semi-automatic gun seems to be completely normal. The gun's slide and chamber are closed as if there are still bullets in the magazine or at least one bullet is chambered. But then the camera focuses on his gun and we see the gun's chamber is open - as if there are no bullets left. The gun didn't fire between shots so it's chamber should have been open in the first place.
Deliberate mistake: During the scene where Carlos and L.J. are searching the motel, L.J. sees a zombie which turns out to be a reflection in a wall-length mirror. L.J. shoots, breaking the mirror and realizing his mistake. But, if he had been standing in front of the mirror as shown, he should have seen his own reflection standing there as well. This was done as an homage to a similar encounter in the original game.
Plot hole: In the beginning of the movie, we see a zombie eating from a corpse in the middle of the road. It then gets hit by our heroes' trucks. What doesn't make sense is the fact that zombie blood (highly infectious) splashes all over the side of the (windowless) bus with kids in it. There's nothing stopping the blood from getting in the kids' faces, yet nobody seems to be alarmed by this.
Other mistake: In the scene where Dr. Isaacs is in the teleconference with the Umbrella Board of Directors, there are several glasses of water sitting in front of each individual hologram. At the end of the teleconference, the holograms of the board members disappear, but none of the glasses follow suit. This tells us that the glasses of water were physically in the room with Dr. Isaacs, for absolutely no reason, given all the other attendees weren't really there.
Plot hole: In the scene where Alice breaks into the Umbrella compound she drives the vehicle through the truck wreckage and into the fence, knocking the fence down. However, she then has time to ferry everyone from the vehicle into the chopper, stand back and watch them fly away, then walk cautiously into the shanty shack and wait for the elevator to arrive. Meanwhile not a single zombie has chased her into the now breached compound. The zombies might be dumb, but if they see food they head straight to it until an obstacle stops them and the explosion would have only killed the ones in the immediate area. The ones behind the dead zombies would have charged at them as soon as the fire died down.
Plot hole: When the survivors arrive in Las Vegas, all that is visible is the Strip. No other buildings are seen, even though Las Vegas has other high-rise buildings that are not on the Strip, and the level of sand that has built up is not substantial enough to have buried the entire city.
Continuity mistake: In the scene where Dr. Isaacs escapes from Alice in a AS350B Eurocopter, we see that the helicopter's landing gear has an additional iron bar that has been changed from one scene to another. When the helicopter lands in the desert, the bar is missing. So Dr. Isaacs flew away in one helicopter and landed in another.
Other mistake: In the end fight scene between Dr. Isaacs and Alice, she bumps into the water cocoon and awakes one of her clones only to have it die a few seconds later. When she goes to cover up the body, the clone can be seen wearing a wedding ring - they don't incubate the bodies wearing jewelry in an embryonic state.
Other mistake: In order to use the vials that have either the T-virus or the anti-virus it has to be loaded into a special gun-like tool, similar to a caulking gun. A plunger is then set, and released with a trigger, again, like a caulking gun. The problem is that when the vials are seen, the caps on either end are solid, with no hint of an opening for the fluid to get out and into the body that it is being administered to.
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Revealing mistake: After Carlos blows his truck up and Alice is breaking into the Umbrella compound there is a moment where her truck drives through the fire and one of the zombies following her throws his arms up to shield himself from the heat of the flames. The actor apparently got too close for his own comfort.