Today's action comes as US officials point to new evidence that Russia is amassing new weapons and munitions closer to its border with Ukraine.
Russia had pulled back its troops from the border in recent weeks as negotiators from Moscow and Kiev began sketching out a peace plan.
The new sanctions freeze any assets that the separatists have in the United States, and prohibit any American firms or businesses from dealing with them.
The US has already issued similar sanctions against Ukraine separatists and Russians.
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