Metaphase karyotypes of 2 and 3 species of the Umbrosus and the Barbirostris Groups, respectively, of the subgenus Anopheles occurring in Thailand and Indonesia show inter- and intraspecific differences with respect to the amount and distribution of constitutive heterochromatin in the sex chromosomes and/or autosomes. Four forms of metaphase karyotypes have been recognized in the wild samples of Anopheles barbirostris based on differences in size and shape of X and Y chromosomes. It is not known whether forms A, B, and C of the metaphase karyotype of An. barbirostris found in Thailand represent inter- or intraspecific differences. However, form D, which occurs only in Indonesia, may represent a mitotic karyotype of a distinct species closely related to An. barbirostris. Anopheles campestris and An. barbumbrosus are each readily separated from An. barbirostris by mitotic chromosomes. Anopheles umbrosus and An. letifer of the Umbrosus Group also exhibit heterochromatin variation in the X chromosomes. These 2 species can be readily distinguished by the gross morphology of mitotic karyotypes, particularly the X and Y chromosomes.