Police spokeswoman Lucie Novakova says the crash happened early today afternoon near the town of Melnik, north of Prague.
Novakova says that of the six Americans traveling in a rented Mitsubishi Outlander, two of them a man and a woman died immediately. A woman died in a hospital in the northern city of Liberec.
Another woman from the US was transported in critical condition to Prague's Military University Hospital, while two men escaped unharmed.
Police would not reveal the identity of the victims. Novakova says police are investigating, but a possible cause might have been that the car didn't give the right of way to the truck.