One airport employee said the aircraft exploded in flight, but experts at the crash site said the aircraft caught fire on impact.
Jan Padourek, first secretary at the Czech embassy in Kiev, told Czech television the five victims were Czech citizens.
One witness said there was a destroyed barn in the middle of the field and that sections of a wing, engine and some seats were scattered in the area.
Police and emergency workers were present, he said.
"Five people died," the emergency ministry spokesman said.
"There were five people on board - two pilots and three passengers.
"This was a small plane which was flying to Ukraine and three kilometres from Zhulyany it dropped off the radar screens," he said.
The plane was a Beechcraft B9L.