Electrophoretic studies of isozymes were done with four closely related species of the Simulium perflavum species group (Diptera: Simuliidade) in the Brazilian Amazon, using last-instar larvae collected in the field. Ten enzymes were studied, which yielded 11 loci. Diagnostic loci were not found between Simulium maroniense cytotype D and Simulium rorotaense. Simulium maroniense and S. rorotaense differed from Simulium trombetense by two diagnostic loci (Me and Xdh), and Simulium perflavum differed from the other three species by four diagnostic loci (Me, Xdh, Mdh, and Got). The mean number of alleles per locus ranged from 1.30 to 2.30, the percentage of polymorphic loci ranged from 18.2 to 63.6% and the mean heterozygosity values observed ranged from 0.062 to 0.108. Genetic distances among the species ranged from 0.010 to 0.581. The lowest value was obtained between S. maroniense and S. rorotaense, and the highest between S. perflavum and S. trombetense. The genetic relationships among the four S. perflavum group species indicate that they are closely related. The high similarity at the isozyme level, allied to previous studies of morphology and polytene chromosomes, may suggest that the divergence time since the separation of S. maroniense and S. rorotaense is still too recent for diagnostic loci to have evolved.