Isozyme electrophoresis studies were conduced on Anopheles oswaldoi sensu lato from three states in Amazonian (Brazil), including Acre, where this taxon has been incriminated as a potential human malaria vector. The 13 enzymes analyzed yielded a total of 20 loci. Of these, 10 were monomorphic in the three samples. Diagnostic loci were not found. The measures of genetic variability showed mean number of alleles per locus between 1.5 and 2.0, percentage of polymorphic loci of 40%, and mean heterozygosities from 0.031 to 0.062. The inbreeding coefficient F_IS had a moderate mean value (0.109), which resulted from heterozygote genotype deficiencies and the presence of rare alleles in the homozygote state. Mean F_ST value (0.0502) and genetic distance values (Nei’s genetic similarity values of 0.994–0.997; Roger’s genetic similarity values of 0.963–0.968) were very low among the three samples of An. oswaldoi s. l. Preliminary results of identifications of male genitalia indicated that An. oswaldoi s. s. Peryassú and Anopheles konderi Galvão & Damasceno of the An. oswaldoi complex coexist sympatrically in Sena Madureira, Acre, and São Miguel, Rondônia, whereas in the sample from Coari, Amazonas, only An. konderi was identified. The isozyme results do not suggest differences between these species, particularly in areas of sympatry.