Isofemale cultures of wild-caught Anopheles culicifacies collected from 11 localities representing different ecoepidemiological zones on the mainland of India were identified by examining both F1 male larval mitotic karyotypes and polytene chromosomes of half-gravid F1 adult females. All cultures identified as species A by polytene chromosome examination had submetacentric Y chromosomes. In species B and C, some isofemale cultures had acrocentric Y chromosomes, whereas others were submetacentric. The study revealed the existence of a Y chromosome polymorphism in species B and C; consequently, male mitotic karyotypes are of limited use for differentiating members of the An. culicifacies complex.