Historically, the taxonomy and nomenclature of Japanese salmon have been in a state of confusion. Masu, amago and biwa salmon have been variously classified as distinct species, subspecies, or often conflicting or overlapping combinations of the two. In particular, the taxonomy of masu and amago salmon is obscured by their similarity in ecological and morphological traits. Here, DNA sequence analyses of nuclear and mitochondrial DNA were applied to clarify the genetic relationship between masu and amago salmon. No fixed differences were detected in the mitochondrial ND3 gene and control (D-loop) region, or in the nuclear growth hormone type-2 gene (GH2). However, the frequency of single nucleotide substitution alleles within GHZ intron C and size variants at a microsatellite locus nested within intron D differed markedly, providing genetic evidence to support a taxonomic distinction between the two types. The genetic data were related to previous mitochondrial DNA sequence analyses and alternative classification schemes for masu and amago salmon. The best-supported scheme arranges the two types as subspecies; masu as Oncorhynchus masou masou Brevoort and amago as Oncorhynchus masou ishikawae Jordan and McGregor.