The genetic relationships among 17 species of Astyanax from the Iguaçu River and adjacent river basins in Brazil were examined using nucleotide sequences of the mitochondrial cytochrome b gene (cytb). Congruent trees were constructed using neighbor-joining, maximum-likelihood, and Bayesian methods. The resulting clades suggest that at least three major groups share similar origins with the endemic species of the Iguaçu River. The results indicate that Astyanax is polyphyletic in this location, which suggests that Astyanax did not diversify from a single ancestral group that was isolated when the Iguaçu River basin formed. Astyanax bifasciatus shares an origin with some species of the altiparanae-bimaculatus complex, while A. minor originated from the same group as A. aff. paranae, A. fasciatus, and A. bockmanni. The third group includes A. dissimilis, Astyanax sp F, and A. serratus that are endemic species to the Iguaçu River basin. Geological and hydrological events that influenced the biogeographical patterns of these species are discussed.