State Department spokeswoman Jen Psaki said the vote organized by pro-Russian elements in Donetsk and Lugansk "was an attempt to create further division and disorder in the country."
"We do not recognise the illegal referendum," she said. "It was illegal under Ukraine law."
Pro-Russian separatists claimed a massive victory in the two east Ukraine regions, while Kiev called the balloting a farce.
Psaki added that the "methodology was also highly suspect, with reports of ... Pre-marked ballots, children voting, voting for people who were absent, and even voting in Moscow and St Petersburg."
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The United States and its allies remain focused on the presidential elections organized by the interim government in Kiev for May 25.
"They're very much on track with preparations for the elections," Psaki told reporters, adding that there were already a number of monitors on the ground.
She also denounced threats from the Russian company Gazprom to cut off gas supplies to Ukraine because of debts totalling USD 3.51 billion (2.55 billion euros).
"Obviously, we don't think... Access to energy should be used as a threatening tool," Psaki said.