In situ hybridization of biotin-labeled mouse major satellite DNA clone pMR196 was applied to Mus domesticus and Mus spretus chromosomes (Chr). The same karyotypes were counterstained with distamycin A-DAPI to identify AT-rich heterochromatin. Chromosomes from the laboratory mouse, C57BL/6Ros (BL/6; M. domesticus) were uniformly labeled at the centromere except for the Y, while chromosomes from the divergent Mus species M. spretus showed little or no hybridization. Differences between Mus species in copy number of the major satellite DNA sequence were used to identify chromosomes of M. domesticus and M. spretus in their F1 hybrids and to discriminate domesticus and spretus centromeres in backcross progeny. The distribution pattern of heterochromatic regions demonstrated by distamycin A-DAPI counterstaining was comparable with that of in situ hybridization with pMR196, suggesting that A-T rich heterochromatin in M. domesticus is mainly constructed by the pMR196-related sequence. The in situ technique was used to examine segregation of domesticus centromeres in backcross progeny obtained by mating F1 hybrid females with M. domesticus or M. spretus males. The segregation of centromeres did not deviate from the expected among the backcross progeny from C57BL/6Ros males, whereas chromosomes with M. domesticus centromeres were prone to appear in the progeny from backcross matings by M. spretus males.