Both males and females of the species of spinous country-rats (Tokudaia osimensis osimensis, T. o. o., Rodentia: Muridae), which live on Amami Oshima Island, a southern Japanese island, have 25 chromosomes. Another species of spinous country-rats (Tokudaia osimensis spp., T. o. spp., which live on Tokunoshima Island 40 km south of Amami Oshima Island, also have an odd number of chromosomes, 45. Karyotypes of males and females by the G-band method were indistinguishable in both populations. The lesser number of chromosomes (25) of T. o. o. is likely to be a result of Robertsonian fusions of 45 chromosomes of T. o. spp. that seem to be the offspring of another spinous country-rat Tokudaia osimensis muenninki (T. o. m.), which live on Okinawa Island and have 44 chromosomes including the X and Y Chrs. The lengths of the non-paired, putative X-Chr of T. o. o. and T. o. spp. occupied roughly 3.2% and 1.7% of the total lengths, respectively, hinting at translocation or exchange of a part of the X Chr and thus in violation of Ohno's Law. Southern blot analysis with murine Sry as a probe indicated that these two animals do not have Sry. When Zfx from T. o. spp. was used as a probe, both males and females of T. o. o. and T. o. spp. showed two bands, suggesting possible translocation of Zfy from the Y Chr. Comparison of physical characteristics, constituents of chromosomes, and sex-determination methods of these three Tokudaia country-rat populations suggests that each is endemic to each island and constitutes an independent species. These specialized species would provide us with clues to elucidate the mechanisms of primary sex determination and karyotype evolution in mammals.