Interior Minister Vít Rakusan said the crash took place late Wednesday night in the city of Pardubice, about 100km east of Prague. The high-speed passenger train belonged to the private RegioJet company.
Rakusan said none of the injured was in life-threatening condition.
Rescuers said 380 passengers were on board the train heading for the city of Kosice in eastern Slovakia and further to Chop across the border in Ukraine.
The dead were not immediately identified. The drivers of both trains survived, the local CTK news agency said.
Transport Minister Martin Kupka said the main track between Prague and the eastern part of the country had to be closed while authorities investigated the collision.
It was only partially reopened nine hours later and the state-run train company, Czech Railways, advised that passengers should avoid using the route for the whole day.
The corridor in Pardubice, where the trains collided, is vital for Czech Railways. It said the line will also likely be closed on Thursday.
"We can't and won't speculate about the cause of the accident," Kupka said.
Police said later they were investigating possible negligence.
Prime Minister Petr Fiala called the crash a big tragedy and offered his condolences to the families of those killed.
So did Radim Jančura, the owner of RegioJet, who said his company was ready to compensate the passengers.
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