Mus musculus (M. m.) molossinus has been considered an independent subspecies of Mus musculus. To elucidate the evolutional origin of this subspecies, we carried out double-color FISH using 18s-28s ribosomal DNA and mouse chromosome paint probes. Among eleven rDNA loci detected, five loci on chromosomes 12, 15, 16, 18 and 19 were common to both Mus musculus (M. m.) musculus and M. m. molossinus and the other six loci, on chromosomes 1, 5, 10, 11, 13 and 17, were characteristic in M. m. molossinus. As M. m. molossinus is thought to originate from a hybrid between ancestral colonies of M. m. musculus and Mus musculus castaneus, we supposed that these six rDNA loci might have evolved after geographical isolation of the ancestral hybrid animals from M. m. musculus and M. m. castaneus.