Abstract Simulium goeldii Cerqueira & Nunes de Mello, 1967 and Simulium ulyssesi (Py-Daniel & Coscarón, 2001) share larval and pupal microhabitats in Central Amazonia and are morphologically similar, differing only in the number of gill filaments - six in S. ulyssesi and eight in S. goeldii. To test the hypothesis that S. ulyssesi is a valid species, rather than an intraspecific variant of S. goeldii, we compared the polytene chromosomes of the two morphotypes from Presidente Figueiredo, Manaus, and Careiro da Várzea (Amazonas) Counties. Our analyses indicated that the two morphotypes are distinct species, differing by four fixed inversions. Because the number of gill filaments is correlated absolutely with chromosomal banding patterns, it is a useful structural character for differentiating the two species.