The new list took aim at a key unit of Bank Rossiya, often called the personal bank of Russian president Vladimir Putin, and some of Russia's largest construction companies.
It included 17 Ukrainian separatists, 11 of them officials of the government Russia established in Crimea after illegally annexing the northern Black Sea peninsula in 2014.
And it also blacklisted a large number of Russian companies operating in Crimea, including major maritime and defense businesses.
That would impact not only US banks but also the US branches of foreign banks, making it difficult for them to serve the sanctioned firms.
The US Treasury said the new move aims at countering efforts by Russian entities to circumvent existing sanctions on individuals and companies.
The Treasury stressed the need for Russia to abide by its commitments to the 2014 Minsk agreements aimed at halting the fighting between Ukraine forces and pro-Moscow rebels in eastern Ukraine.
"Russia continues to provoke instability in eastern Ukraine despite its Minsk commitments," said John Smith, the acting director of the Treasury's Office of Foreign Assets Control.
"Treasury stands with our partners in condemning Russia's violation of international law, and we will continue to sanction those who threaten Ukraine's peace, security, and sovereignty."
Newly blacklisted was CJSC ABR Management, an asset management firm closely linked to already-sanctioned Bank Rossiya, which serves members of Putin's closest circle.
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