Genetic variability and divergence were estimated for populations of Anopheles rangeli Gabaldón, Cova-Garcia & Lopes, Anopheles nuneztovari Gabaldón cytotype A, and Anopheles dunhami Causey from the Brazilian Amazon using isozyme electrophoresis. These species are included in the Oswaldoi subgroup, subgenus Nyssorhynchus (Diptera: Culicidae). Thirteen enzymes yielded a total of 22 loci, of which 14 were monomorphic in the three species. Three diagnostic loci (Gpi-1, Hk-1, and Me) and a strong differentiation in the Mdh locus were found between An. rangeli and An. nuneztovari. Five diagnostic loci (Mdh, Gpi-1, Hk-1, Gpd, and Me) separated An. rangeli from An. dunhami, whereas one diagnostic locus (Gpd) separated An. nuneztovari from An. dunhami. Moderate differentiation was observed in the Est-5 and Pgm loci between An. rangeli and An. nuneztovari, and between An. nuneztovari and An. dunhami. Anopheles dunhami had the highest values for three indices of genetic variability, whereas An. rangeli showed the lowest values for mean number of alleles per locus and mean heterozygosity. Nei’s genetic distance was highest between An. rangeli and An. dunhami (0.280) and lowest between An. nuneztovari and An. dunhami (0.072). Between An. rangeli and An. nuneztovari the genetic distance was 0.237. Anopheles dunhami and An. nuneztovari are sister species very closely related and may have a recent evolutionary origin. Anopheles rangeli probably diverged before the other two species separated. This is the first record of An. dunhami in Coari (Amazonas, Brazil) and only the third collection site of this species since its description in 1945.