Anopheles culicifacies sensu lato collected from Rameshwaram island, Tamil Nadu state, India was identified as species B based on the diagnostic inversion karyotype Xab 2g1+h1 as observed in polytene chromosomes. Among male mitotic karyotypes made from larval neurogonial cells, two types were observed: one with an acrocentric Y-chromosome and the other with a sub-metacentric Y-chromosome, both had sub-metacentric X and metacentric autosomes. The Rameshwaram population is identical to species B in its genetic relationship with species A and C as determined by experimental hybridizations (sterile and fertile male hybrids, respectively).