Plot hole: Det. Kerry says at the scene of Paul's trap, "He had two hours." There is no way she could have known that. The clock simply said 3:00 and the tape specifically says "you have until 3 o'clock or this room will become your tomb". No way to know that's two hours after the fact. (00:17:30)
Plot hole: It is said that Drake has been sleeping for 4,000 years. But Drake specifically mentions that he witnessed Christ being crucified, which happened around 2,000 years ago. This harkens back to a plot line where Drake had only been sleeping since the turn of the century, abandoned in favor of the one shown in the movie.
Plot hole: During the end of the film chase scene, Agent Mackelway calls Agent Kulok and gives her a vague location of where they are and asks for back up. Considering Mackelway had been kidnapped and driven out into the country, it should have taken Kulok much longer to arrive at the scene instead of the few minutes that are indicated.
Plot hole: When Ana is driving away from her home, she stops on the road and a guy opens her door, scaring her into turning her car toward the curb, plowing through a guardrail. She couldn't have picked up a lot of speed, and those guardrails are supposed to always be reinforced on highways/interstates. At that angle she would have had to knock out at least 4 reinforcing beams (they use 'I' beams to reinforce them, and are intended to stop vehicles from passing through them, or slow them down immensely).
Plot hole: After the police discover the plot to kill Lynn by her ex-husband, they never bother to visit his house to arrest him. If they did, they would have found him dead already and would have KNOWN that the killer was still at large.
Plot hole: After Dr. Ashford was shot, he stayed down the whole time that Alice fought with Nemesis, they fought with Umbrella, and after they took off. How is that possible? According to all the other times they've been shot, and with the t-virus in their bloodstream, they reanimate almost ten minutes later -- not when the director wants.
Plot hole: When the fat guy throws a boot into the ice-spray room, it freezes and shatters on the ground. You can see the length of the shoelace on the ground as well. The shot cuts to the guy in the shaft, and he pulls up the shoelace to reveal the end encased in ice, while the end is halfway into the ice block. This wouldn't be possible, because the other end of the shoelace had no ice at all, and there's no sign either of it breaking.