The African Anopheles funestus and the Oriental An. minimus groups are closely related and composed of major malaria vectors in Africa and Southeast Asia, respectively. None of the species of either the An. funestus or the An. minimus group can be identified with absolute certainty using the adult morphology. Polymorphisms present on the internal transcribed spacer 2 (ITS2) of ribosomal DNA allowed the development of 10 primers that combined with an universal forward primer lead to a simple and sensitive multiplex allele-specific polymerase chain reaction (AS-PCR). Moreover, the possible additional amplification of the entire ITS2 allows one to detect other anopheline species in sympatry with members of both groups not included in this assay and serves as a control band. This universal PCR method permits the discrimination of 10 species within the subgenus Cellia, among which figure three major malaria vectors, and constitutes a very efficient and powerful tool to improve our knowledge on these species distribution and biology. Not only restricted to anophelines, this AS-PCR could also be developed and applied to other insect groups.