Recent polls showed a large majority of around 70 per cent in favour of staying part of Canada, while roughly 20 per cent supported secession, with the remainder undecided.
Alberta Premier Danielle Smith, Canadian Prime Minister Mark Carney and both of the country's major political parties have repeatedly spoken out against secession.
By contrast, support for the independence movement has come from the administration of US President Donald Trump.
Alberta, a province of around five million people, has some 3.1 million eligible voters, according to Elections Alberta, the provincial election authority.
Polls opened at 9am on Monday (2am Tuesday AEDT) and close at 6pm (11pm AEDT) though postal and in-person advance voting has already taken place.
Voters will decide on a total of 10 questions, with the secession question to be counted first. A result could be available as early as Tuesday, according to election officials.
Calgary, with around 1.68 million residents, is Alberta's largest city, followed by the provincial capital of Edmonton with 1.28 million. The province is also known for its national parks in the Rocky Mountains, which attract tourists from around the world.
Alberta has some of Canada's most conservative voters, and a movement advocating independence on cultural and economic grounds has existed for years.
Some oil industry representatives feel restricted by environmental regulations, while many protesters who opposed coronavirus restrictions and vaccine mandates later joined the independence movement.
Although the movement has long represented a minority of Albertans, it succeeded in persuading Smith, the province's conservative premier, to call the referendum. Smith herself has consistently advocated remaining part of Canada.
"Canada works," he said during a visit to the province last week.
"We have to be strong and we are stronger together," Carney said.
"Generations of Albertans helped build this country. It's yours, it's ours, and it's worth fighting for."
The referendum comes amid an escalating US-Canada trade dispute, with Trump repeatedly threatening Canada and floating the idea of making it the 51st US state. Carney has urged
