Multilocus enzyme analysis of the genetic variability and population structure was conducted among three malaria vector species of the Anopheles nili group in Cameroon: An. nili, An. carnevalei, and the recently described An. ovengensis. We detected species-specific alleles and large differences in shared allele frequencies at six of nine loci (e.g., PGM, GOT1, IDH1, IDH2, PGI, and alpha-GPD). This non-random distribution of alleles leads to high and significant values of differentiation indexes (0.569 < Fst < 0.874, P < 10(-4)). These results fully agree with standard morphologic descriptions, and therefore provide further support for recent taxonomic classification within the An. nili group.