Police said the crash occurred shortly after 8am (3pm AEST) in the town of Milavce.
Czech Railways said an international high-speed train that connects the western Czech city of Plzen with Munich in Germany and a local passenger train were involved in the collision.
The regional fire department said at least two people have died.
Dozens of people were injured, five of them seriously, the Fire Rescue Service of the Plzen region said in a statement. Four helicopters were transporting the injured to nearby hospitals.
Czech Transport Minister Karel Havlicek said human error likely caused the crash. He said the driver of the high-speed train had failed to stop at a designated location.
“The situation is serious,” said Havlicek, who was heading to the site.
