Metaphase karyotypes of eight species of the Maculatus Group exhibit inter- and intraspecific variation based on differences in the amount and distribution of constitutive heterochromatin in the sex chromosomes and/or in the centromeric regions of the autosome(s). Analysis of heterochromatic variation has revealed three distinct groups of mitotic sex chromosomes. Anopheles sawadwongporni, An. dravidicus, and An. pseudowillmori form Group 1, which is characterized by telocentric or acrocentric sex chromosomes. The only representative of Group 2 is An. notanandai, which has a metaphase karyotype showing a unique chromosome X with a very small amount of centromeric heterochromatin. The remaining four species studied (An. dispar, An. maculatus, An. willmori, and An. greeni) exhibit a third group of sex chromosomes showing subtelocentric (acrocentric), submetacentric, or metacentric configurations.