Karyotypes and crossing relationships were investigated for three allopatric populations of Anopheles leucosphyrus in Southeast Asia: South Kalimantan, Sumatra and Thailand. The mitotic karyotypes of these populations were similar to those previously observed in other species of the An. leucosphyrus group. Populations from Thailand and South Kalimantan exhibited telocentric and subtelocentric sex chromosomes, respectively, with a distinctive band of intercalary heterochromatin in the X chromosome. Strikingly different submetacentric X and Y chromosomes were observed in the population from Sumatra, and it seems likely that the evolution of these chromosomes occurred through the acquisition of constitutive heterochromatin. Sterile F1 males were observed in crosses between the Sumatra population and the populations from South Kalimantan and Thailand. No genetic incompatibility was observed in crosses between the latter two populations. We believe that the present concept of An. leucosphyrus includes two allopatric species, one inhabiting Borneo, West Malaysia and southern Thailand and one confined to Sumatra.