Susan Rice, President Barack Obama's national security adviser said it is not in the interests of Ukraine, Russia, Europe or the United States to see the country split.
Speaking on the NBC program 'Meet the Press', Ms Rice was asked about a possible scenario in which Russia would send forces into Ukraine to restore a government more friendly to Moscow.
She said: 'That would be a grave mistake. It is not in the interests of Ukraine or of Russia or of Europe or the United States to see the country split. It's in nobody's interest to see violence return and the situation escalate.'
Ms Rice's comments are the most extensive from the White House to date since the recent dramatic events unfolded in Ukraine.
It comes as Ukraine's new interim president said on Sunday that he was open to dialogue with Russia as long as Moscow respected his country's decision to seek closer ties with the European Union.
'We are ready for a dialogue with Russia... that takes into account Ukraine's European choice, which I hope will be confirmed in (presidential) elections' set for May 25, Oleksandr Turchynov said in a television address.
As the Winter Olympic Games in Sochi, Russia, end Putin is now likely to more closely focus on the Ukrainian situation.
The State Department said on Saturday it would send its No. 2 official, Bill Burns, to Kiev this week.
Rice said the United States wants a de-escalation of the violence in Ukraine, constitutional changes, democratic elections 'in very short order,' and the opportunity for Ukrainians to come together in a coalition unity government.
She said U.S. authorities are not sure where Russian-backed President Viktor Yanukovich has fled.
'He is in a place that will reveal itself. Yesterday we knew where he was. Today we're not so sure,' Rice said.
The crisis in Ukraine reflects the conflict between those who want the country to remain aligned with Moscow and Russian President Vladimir Putin, and those seeking closer integration with Western Europe.
But Rice said those goals were not 'mutually exclusive'.
'There is not an inherent contradiction ... between a Ukraine that has longstanding historic and cultural ties to Russia and a modern Ukraine that wants to integrate more closely with Europe,' Rice said.
On Sunday, Ukraine's parliament, exercising power since mass protests caused Yanukovich to flee, named its new speaker as acting head of state and worked to form a new government.
The European Union and Russia, vying for influence over the huge former Soviet republic on their borders, considered their next moves.
Russia, which had provided funding to Yanukovich's government, said it would keep cash on hold until it sees who is in charge.
Asked whether Putin looked at Russia's sphere of influence in Ukraine in a Cold War context, Rice said that 'he may'.
'But if he does, that's a pretty dated perspective that doesn't reflect where the people of Ukraine are coming from. This is not about the U.S. and Russia,' Rice added.
Two key lawmakers urged the Obama administration to make clear that Ukraine's territorial integrity must be protected.
'I think the message has to be sent to him (Putin) that let the Ukrainian people determine their own future, and a partition of Ukraine ... is totally unacceptable,' Republican Senator John McCain, a key Republican voice on foreign policy, told the CBS program 'Face the Nation'.
'And we need to act immediately to give them (Ukrainians) the economic assistance that they need, based on reforms that are gonna be required, as well. So it's gonna be tough sledding.'
Bob Corker, the top Republican on the Senate Foreign Relations Committee, added in a statement: 'The United States should do everything possible to ensure Ukraine remains one country and that their territorial and political integrity is maintained, allowing them the freedom to choose a future within Europe.'
Earlier on Sunday, U.S. Treasury Secretary Jack Lew met his Russian counterpart, Anton Siluanov, on the sidelines of a Group of 20 meeting in Sydney to discuss the situation in Ukraine.
'Secretary Lew emphasised that the United States, working with other countries including Russia, stands ready to assist Ukraine as it implements reforms to restore economic stability and seeks to return to a path of democracy and growth,' a Treasury Department official said.
Earlier today, images emerged of former Ukrainian Prime Minister Yulia Tymoshenko in tears as she was reunited with her daughter for the first time in two and a half years after she was released from prison.
In emotional scenes the opposition politician was captured kissing her daughter Yevgenia upon her arrival to Kiev's iconic Independence Square to address the assembled crowd.
With its president gone and a new one named today to fill the void, Ukraine remains a country bitterly divided by an ethnic split that goes back to the collapse of the Soviet Union two decades ago.
For three months anti-government protesters have been involved in a stand-off with authorities.
With the news that President Viktor Yanukovich has fled Kiev and former prime minister Yulia Tymoshenko has been released, the nation's future hangs in the balance.
Comment: After such a deliberate and intense effort to overthrow the Ukrainian government and engineer this crisis in the first place, that US officials can spout such nonsense is just laughable.