Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.