Three arrangements of chromosome arm 2 occur as homozygotes, without observable heterozygotes, in populations of Anopheles fluviatilis James. This indicates positive assortative mating between populations having different arm 2 arrangements and is taken as evidence for three sibling species within A. fluviatilis . This finding confirms an earlier hypothesis that the taxon is a mixture of biological forms, which was indicated by heterogeneity between populations in their malaria transmission potential on the Indian subcontinent. Rearrangement of arm 2 involves two paracentric inversions, 2q1 and 2r1 giving three types of arm 2: 2 + q1 + r1, 2q1 + r1, and 2 + q1r1. Photomaps of the ovarian polytene chromosomes are presented.