Anopheles quadriannulatus Theobald historically has been reported from southern Africa, Zanzibar islands, and Ethiopia. However, based on evidences of genetic incompatibility between crosses of South African and Ethiopian populations, the population from Ethiopia was recently reported as a distinct species designated as An. quadriannulatus sp. B. An. quadriannulatus sp. A, denoted the southern African population. To distinguish the two populations, the IGS (intergenic spacer) region of rDNA was sequenced to design a primer specific for An. quadriannulatus sp. B. A cocktail polymerase chain reaction (PCR) involving Anopheles gambiae Giles universal (UN) primer, the new primer and other primers specific for members of the An. gambiae complex produced the expected diagnostic products for the respective species. Using extracted DNA and crushed body parts as sources of template DNA, this assay was reliably used to identify samples of An. quadriannulatus sp. B.