Obama, in a statement to White House reporters, alleged that Russia-backed Ukrainian separatists are denying access to the crash site to the team of international investigators including those from the US.
He also accused the separatists of destroying crucial evidence from the crash site including removal of bodies.
"Separatists are removing evidence from the crash site. All of which begs the question, what are they trying to hide?" asked the US president.
"What they need right now is immediate and full access to the crash site," he said.
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Noting that Russia has influence over the Ukrainian separatists, Obama urged Putin to ask them to cooperate fully in the investigation.
"Given its direct influence over the separatists, Russia has direct responsibility to encourage them to stop tampering with the evidence," said Obama.
"Now's the time for President Putin and Russia to pivot away from the strategy that they've been taking and get serious about trying to resolve hostilities within Ukraine," Obama said. "And time is of the essence," he added.