Anopheles subpictus Grassi is shown to comprise four reproductively distinct species, designated A, B, C and D, occurring sympatrically in villages of Pondicherry, southeast India. Adult females were reared individually from wild larvae and examined for their morphological and chromosomal characters. Paracentric fixed inversions on the X-chromosome serve to distinguish the species cytogenetically, with no inversion heterozygotes (i.e. no interspecific hybrids) among totals of 717 species A (X+a, +b), 1863 species B (Xa, b), 869 species C (Xa, +b) and 1365 species D (X+a, b) identified. Morphologically, diagnostic characters for each of the four species are seen in the egg float ridge number, larval mesothoracic seta 4, pupal seta 7-I and the palpi of female adults. Species A, C and D immatures inhabit freshwater, whereas the malaria vector species B breeds in saltwater and was found only in coastal villages.