The expulsions were unveiled after the Czech ambassador in Moscow, Vitezslav Pivonka, had been summoned by the Russian foreign ministry.
"Pivonka had been told that 20 employees of the Czech embassy in Moscow had been declared 'persona non grata'. They have been ordered to leave our country by the end of April 19, 2021," the foreign ministry said in a statement.
The ministry also demanded that the number of locally hired staffers at the Czech Embassy be reduced to the same level as that of the Russian Embassy in Prague.
On Saturday, Czech Prime Minister Andrej Babis announced that 18 Russian diplomats had been declared persona non grata over their alleged links to huge explosions in 2014 at military warehouses in the town of Vrbetice which left two people dead.
The diplomats were asked to leave the country within 48 hours.
Russian Foreign Ministry spokeswoman Maria Zakharova called the decision of the Czech authorities a "trick" and promised a response from Moscow.
"Prague is well aware what follows such ‘tricks’," she said.