The new regulation under the Ontario Heritage Act applies to the Edmund Fitzgerald shipwreck in Lake Superior, one of the best known shipwrecks in the Great Lakes.
It also affects the Hamilton and Scourge in Lake Ontario.
Culture Minister Madeleine Meilleur says anyone wishing to dive to one of these sites or to operate research equipment near them will require a licence from the province.
Meilleur says the sites need special protection because they contain human remains and must be treated with care and respect.
The Edmund Fitzgerald was an American bulk carrier that sank in a storm Nov. 10, 1975.
All 29 crew were lost in Canadian waters northwest of Whitefish Point, Mich.
The Hamilton and Scourge were merchant schooners pressed into naval service during the War of 1812.
Both sank in Lake Ontario, north of Port Dalhousie, in August 1813. Of the 72 crew aboard both ships, 53 perished, the single greatest loss of life on the Great Lakes during that war.
"The sites we have chosen for special protection are unique,'' said Meilleur.
"We want to ensure that these fragile underwater sites _ all of which contain human remains _ are treated with care and respect.''