It is generally accepted that there are 3 subspecies of Oncorhynchus masou in Japan, namely, Masu salmon (Oncorhynchus masou masou (Brevoort)), Amago salmon (O. masou ishikawae Jordan & McGregor), and Biwa salmon (O. masou rhodurus Jordan & McGregor or O. masou subsp. Kimura). Since the genetic relationship of these three taxa is not well known, there has been considerable confusion over their nomenclature. We have clarified the genetic relationship among these three taxa by partially sequencing their mitochondrial DNA. Sequences of 948 base pairs from the 3' region of the ATPase subunit 6 gene to the 5' region of the cytochrome oxidase subunit 3 gene were obtained for 20 individuals including wild Biwa salmon, wild and farmed Amago and Masu salmon. Furthermore, 2,162 base pairs from the 3' region of ATPase subunit 6 gene to the 5' region of NADH dehydrogenase subunit 4L gene were determined in 4 individuals. In total, there were 26 sites of base substitutions. The haplotypes of Masu salmon and Amago salmon were similar. On the other hand, 17 of the 26 sites had substitutions characteristic of Biwa salmon. A matrix of genetic distances and maximum parsimony analysis among the haplotypes indicated that Biwa salmon is genetically more distant from Masu and Amago salmon, than Masu salmon is from Amago salmon. This means that Biwa salmon diverged from the common ancestor of the Oncorhynchus masou complex before the divergence between Masu salmon and Amago salmon.