Simulium cauchense Floch & Abonnenc and Simulium quadrifidum Lutz are widely distributed in the Amazon region and are morphologically similar at the larval and pupal stages. Chromosomally, these species are readily distinguished by the position of the nucleolar organizer, which is in the short arm of chromosome I in S. cauchense and in the long arm of chromosomes III in S. quadrifidum. They also differ by three fixed inversions. Sex chromosomes are undifferentiated in both species. Chromosomal resolution of the two species allowed us to evaluate four structural features previously used as diagnostic aids at the larval stage. Characters that distinguish larvae of the two species are the number of branches and branching patterns of the dorsal abdominal setae and the dark band on each primary fan. Branching patterns of the gill histoblasts were often diagnostic, with S. quadrifidum exhibiting more proximal branching and S. cauchense more distal branching. Sites where both species occurred sometimes had larvae with one petiole branching proximally and the other distally; in these cases examination of the chromosomes permitted assignment of the specimen to species. Pigmentation patterns of larvae, on the other hand, are highly variable. Color typically is sex linked in both species