Cytogenetic observations on an extensive material of the Anopheles culicifacies complex from the Thenpennai river area, Tamil Nadu, South India show that the populations are made up of four cytologically distinct species. Three of these, labeled A, B and C have already been described. A fourth one, monomorphic for the inversion arrangement +a +b in the X chromosome and il + hl in the second chromosome, is designated as D, another morphologically indistinguishable sibling species. Each sibling species is characterized by a species-specific type of Y-chromosome. Both of these criteria, inversions and Y-chromosomes can be used to distinguish these important malaria vectors.