Cytogenetic and crossing data provide strong evidence for the existence of another species, dirus E in southwestern India, within the Dirus Complex of Anopheles. These findings are in accord with unpublished morphological observations. Our data suggest a significant genetic divergence between species E and its close relatives, An. dirus A, B and C in Thailand. These data also suggest that dirus E is an incipient sibling species of its geographically nearest relative, dirus D, and that it seemingly co-evolved through the process of allopatric speciation.