Eight Ukrainians in leadership positions in the rebel-held Donetsk region of eastern Ukraine are the targets of the new sanctions, as are armed separatist groups known as the Donetsk People's Republic and the Luhansk People's Republic.
Russian arms, financial and energy companies are also facing further Canadian sanctions.
Prime Minister Stephen Harper pointed to the downing of a Malaysian Airlines passenger jet last week over rebel-held territory in eastern Ukraine as proof that Vladimir Putin's continued support of the rebels "constitutes a very real threat to international peace and security."
In a statement on Thursday, Harper accused the two separatist groups of engaging "in egregious acts against the armed forces of Ukraine, international monitors deployed to the area, and the civilian population" with direct support from Russia.
"Canada remains committed to working with our allies and partners in the international community to preserve and promote a free, democratic and peaceful world," he added.
"We are ready for further actions if the Putin regime's military aggression continues."
Ukraine's ambassador to Canada, meantime, appeared to dial back comments he made to the Globe and Mail when he complained that Canada has failed to deliver millions in financial aid that it's promised to the eastern European country.
Vadym Prystaiko was quoted as saying that he was "at the end of my patience" with Canada.
But on Thursday, his office issued a statement saying he was "grateful for Canada's commitment, the support Canada has provided and Prime Minister Harper's strong stand which has led the international response to the crisis facing Ukraine."
Canada had pledged Ukraine more than $200 million in loans and loan guarantees, as well as communications devices, medical supplies and military equipment that includes body armour. The ambassador told the Globe neither the money nor the gear has arrived, although the Department of Foreign Affairs says it's on its way.
Meanwhile, Canadian sanctions against Russia appear to be affecting Canadian companies.
Canada's Bombardier Inc. says the Canadian sanctions already imposed on Russia could have an impact on the timeline of the company's plans to set up a plant in the country.
The Montreal-based giant was hoping to conclude negotiations this year with Russian company Rostec for the assembly of 100 Q400 regional jets in a project estimated at $3.4 billion.
Company spokeswoman Marianella Delabarrera said this week that Bombardier (TSX:BBD.B) is now being "realistic" about the possibility the project will be ready later than originally expected.
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