These leaders include self-proclaimed rebel mayors, governors and commanders in chief of cities under siege.
The sanctions came as US officials renewed accusations that Russia is providing separatists with tanks and heavy weaponry and as Ukraine's president announced a unilateral ceasefire that Washington urged Moscow to support.
The penalties on seven separatists are a signal that the US will continue to punish those it holds responsible for instability in Ukraine, officials said.
It is hoped they will show the West is still prepared to slap tougher sanctions on Russia's economic sectors to punish it for stoking unrest in Ukraine, should Moscow not take steps to de-escalate the crisis.
The White House welcomed Ukrainian president Petro Poroshenko's ceasefire announcement even as it denounced Russia for supplying separatists with military gear.
White House spokesman Josh Earnest called reports of a Russian military buildup near the Ukraine border “troubling” and dismissed explanations from the Kremlin that the movement of the forces was related to border security.
“We will not accept any use of Russian military forces, under any pretext, in eastern Ukraine,” Earnest said.
He also slammed Russian officials for making what he said were false claims about the Ukrainian government being responsible for poor human rights conditions in eastern Ukraine.
“We see these statements for what they are: an attempt to create pretext for further illegal Russia intervention in Ukraine,” he said.
“Responsibility for the deterioration in the human rights situation in Ukraine lies with the armed separatists who are targeting the population, and their backers in Russia.”
President Barack Obama spoke by phone on Friday to German Chancellor Angela Merkel, who has been a key interlocutor with Russian President Vladimir Putin, and with French President Francois Hollande.
The White House said Russia's failure to take immediate steps to calm tensions would result in further penalties from the US and the European Union — a threat the West has been making for months.
Washington last week accused Russia of sending tanks and rocket launchers to the separatists and State Department spokeswoman Jen Psaki said on Friday that the US believes Russia is preparing to send more tanks and artillery from southwest Russia.
Psaki would not say if the provision of the equipment to the separatists would be a trigger for additional sanctions.
Since Russia annexed Ukraine's Crimean peninsula in March, the US has imposed sanctions against 71 individuals and entities involved.
The European Union has taken similar steps and US and EU officials will meet next week to discuss the possibility of wider sanctions.
The sanctions imposed yesterday freeze any assets that the seven separatists have in the US, and prohibit American firms or businesses from dealing with them.
It is a relatively limited blow against the separatists but shows the US is ready to do more if tensions ratchet back up.
Last month, in an apparent attempt to ease tensions, Putin had pulled back many of his estimated 40,000 Russian troops massed along the border.
They appear to have returned over the last week — even as Putin and Poroshenko discussed the newly elected Ukrainian president’s plan for a unilateral ceasefire and Putin said he was resisted rebel calls for help.