To further understand the phylogenetic relationships among the mustelid genus Mustela, we newly determined nucleotide sequences of the mitochondrial 12S rRNA gene from 11 Eurasian species of Mustela, including the domestic ferret and the American mink. Phylogenetic relationships inferred from the 12S rRNA sequences were similar to those based on previously reported mitochondrial cytochrome b data. Combined analyses of the two genes demonstrated that species of Mustela were divided into two primary clades, named “the small weasel group” and “the large weasel group”, and others. The Japanese weasel (Mustela itatsi) formerly classified as a subspecies of the Siberian weasel (M. sibirica), was genetically well-differentiated from M. sibirica, and the two species clustered with each other. The European mink (M. lutreola) was closely related to “the ferret group” (M. furo, M. putorius, and M. eversmanii). Both the American mink of North America and the back-striped weasel (M. strigidorsa) of Southeast Asia were more closely related to each other than to other species of Mustela, indicating that M. strigidorsa originated from an independent lineage that differs from other Eurasian weasels. Based on biochemical, cytogenetic, and molecular differences as well as morphological evidence, it is proposed that the American mink be elevated to a distinct mustelid genus, Neovison.