Simulium perplexum Shelley, Maia-Herzog, Luna Dias & Couch is structurally similar in the pupal stage to Simulium guianense Wise, the main vector in the onchocerciasis foci of Amazonian Brazil and Venezuela. We report S. perplexum for the first time beyond its type locality (Guyana, Potaro River), describe its larva, redescribe its pupa, and provide a chromosomal comparison with S. guianense and other morphologically similar species. We collected it in two rivers in Rurópolis municipality, Pará state, Brazil. The larvae can be distinguished from those of related species by having body cuticle with microscopic, translucent, and lanceolate setae. Chromosomal comparisons of S. perplexum and similar Brazilian species with available chromosome information (S. guianense, S. hirtipupa Lutz, and S. litobranchium Hamada, Pepinelli, Mattos-Glória & Luz), using S. guianense Cytoform A as the standard, show that S. perplexum has the nucleolar organizer uniquely in the middle of the short arm of chromosome I, whereas the other three species have this marker at the base of the long arm of chromosome I. All chromosome arms, except IIS and IIIS, of S. perplexum are rearranged, compared with S. guianense Cytoform A, suggesting that it is not closely related to this species or to S. litobranchium, as suggested by some authors, based on morphological features.