The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction-site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.