Oleksandr Turchynov told Ukrainian television that roadblocks had been set up around the capital over fears of "provocative" action by covert Russian operatives on May 9, when Ukraine celebrates victory in World War II.
"War is in effect being waged against us, and we must be ready to repel this aggression," said Turchynov, who has placed Ukraine's armed forces on combat alert and reintroduced conscription amid fears of a Russian invasion.
In a last ditch effort to head off a wider confrontation, the chairman of the Organisation for Security and Cooperation in Europe, Didier Burkhalter, was due in Moscow on Wednesday amid calls for his group to mediate between Kiev and the separatists in the east.
But while preparations for his visit were under way, Ukraine's military tightened its stranglehold around the rebel bastion of Slavyansk in the east, cutting off its main road access to the regional capital Donetsk and completely encircling the flashpoint town.
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The violence wracking Ukraine has spread from pro-Russian bastions in the east to the scenic southern port city of Odessa, where dozens of people have died in clashes.
On Sunday, an angry mob of thousands of pro-Russian militants stormed the Odessa police headquarters and forced the release of 67 of their comrades who had been arrested during deadly clashes two days earlier.
Ukraine observed two days of mourning after Friday's violence in Odessa, where running street battles between rival militants culminated in a building fire that killed 38 people. Four others died in gun battles.
The violence was part of a Russian plan "to destroy Ukraine and its statehood," Yatsenyuk charged.
Interior Minister Arsen Avakov said on Monday that 42 of the remaining inmates arrested during the clashes would be moved to other parts to Ukraine to prevent further bids to spring them.
"The Odessa police acted in a disgraceful and possibly criminal way. The police chiefs have been fired. We are investigating their actions.