Revealing mistake: A "green screen" was obviously used at the restaurant (all-you-can-eat buffet). For example, the people supposedly seated in the booth behind Greg's mom were sometimes literally back-to-back with her - there was no back of the booth between them! They were also larger (closer).
Deliberate mistake: For an unknown amount of time during the family's four-day road trip, a Spanish tutorial cassette or CD was played. This would not be enough time for anyone to be able to fluently speak Spanish or carry on a conversation, but toddler Manny was able to do so near the end of the movie.
Visible crew/equipment: As all the stuff is flying out of the boat, you can see the hand of the guy throwing it out.
Continuity mistake: When the family's in the van driving to the hotel, Roderick and Greg switch seats although the van never stops.
Other mistake: When the family got to the "Sleep Tite" motel, the "Beardo family" was in the hot tub. Greg apparently changed from his dark blue or black shorts to white and green swimming trunks when he went into the bathroom. A little later, he saw that the Beardos were returning to their motel room, meaning they were out of the hot tub. Greg already had his swimming trunks on when he asked if he could go to the hot tub. It didn't make sense for him to change as soon as he got to the motel.