During the 16th and 17th centuries, Basque whalers travelled annually to the Strait of Belle Isle and Gulf of St. Lawrence to hunt whales. The hunting that occurred during this period is of primary significance for the North Atlantic right whale, Eubalaena glacialis (Müller, 1776), because it has been interpreted as the largest human-induced reduction of the western North Atlantic population, with ~12250–21 000 whales killed. It has been frequently reported that the Basques targeted two species in this region: the North Atlantic right whale and the bowhead whale, Balaena mysticetus L., 1758. To evaluate this hypothesis and the relative impact of this period of whaling on both species, we collected samples from 364 whale bones during a comprehensive search of Basque whaling ports from the 16th to the 17th century in the Strait of Belle Isle and Gulf of St. Lawrence. Bones were found and sampled at 10 of the 20 sites investigated. DNA was extracted from a subset (n = 218) of these samples. Analysis of the mitochondrial cytochrome b region identified five whale species. The identification of only a single right whale bone and 203 bowhead whale bones from at least 72 individuals indicates that the bowhead whale was likely the principal target of the hunt. These results imply that this whaling had a much greater impact (in terms of numbers of whales removed) on the bowhead whale population than on the western North Atlantic right whale population.