The Glitsiniya property on the Crimean peninsula was bought last year for $US15 million by a Russian bank on behalf of the Kremlin.
Featuring an Olympic-sized indoor swimming pool, huge gardens and a private beach, it was built in 1956 as a summer dacha for Nikita Khrushchev, the former Soviet leader.
It has more recently been used as a sanatorium and requires considerable work to make it suitable for presidential use.
Ukrainian prosecutors have filed a complaint with a court in Kiev, the capital, claiming the villa was illegally privatized, rendering its sale invalid.
The dispute threatens further to sour relations between Putin and Viktor Yushchenko, Ukraine's pro-Western president who came to power after last year's "orange revolution". The Kremlin had backed Yushchenko's rival, Viktor Yanukovich.
"The villa should not have been privatized and sold," said Natalia Boyarkina of the prosecutor's office in Crimea. "According to the law, it cannot belong to anyone other than the state of Ukraine. We have passed the necessary documents to Kiev and are waiting for the courts to act."
The resort was used during the Soviet period by Khrushchev's successors, Leonid Brezhnev, Yuri Andropov and Mikhail Gorbachev.
In Brezhnev's day, the villa even had a rooftop tennis court. It was converted into a restaurant once the Soviet leader's entourage became too frail to play.
The Ukrainian move comes amid attempts by Yushchenko's Government to revisit the privatization of state enterprises and property under Leonid Kuchma, his predecessor.
"A dispute over his holiday home is the last thing Putin needs at a time when he is trying to establish normal relations with Yushchenko," said one Russian diplomat involved in relations with Kiev.
"We have had to swallow our pride over the orange revolution. To have this villa sold to the Kremlin and then taken away is just the sort of petty row that will infuriate him."
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