ABSTRACT.A species complex hypothesis involving Astyanax fasciatus from southern Brazil was tested using 12S mtDNA sequences. Phylogenetic inferences were performed with maximum likelihood, maximum parsimony and Bayesian as phylogenetic methods and Hemigrammus bleheri as the outgroup. Besides 11 sequences from A. fasciatus, the data set was comprised of other partial 12S sequences including material from Astyanax altiparanae (two sequences) and Astyanax sp (four sequences), both from the Iguaçu River. The hypothesis of an A. fasciatus species complex was reinforced given the close relationship between A. altiparanae and Astyanax sp observed in the Bayesian tree. Consequently, a taxonomic revision is necessary for these species.