Astyanax altiparanae, belonging to the bimaculatus group, which includes species with similar colors and morphology, occurs in the upper Paraná River basin. As the use of mitochondrial DNA has made great strides in the diagnosis of species, in previous researches, two strains were detected in A. altiparanae with a high divergence in the D-loop region, provisionally called AltoPR and AltoPR-D. Evidence led to the hypothesis that the two strains did not belong to the same species. Phylogenetic hypotheses were produced by maximum-likelihood. Mean internal distances of the AltoPR and AltoPR-D groups were respectively 0.002 and 0.003, with the distance between them being 0.037. Sequences from GenBank of specimens collected from the Paraíba do Sul River basin were also divided into two groups, of which one may be identified as AltoPR. Since the other group provided an intermediate distance when compared to AltoPR-D, an in-depth investigation was required. The other species analyzed showed a greater distance and was revealed to be a monophyletic taxon. The results suggested that they are really two species and that neither corresponds to the other species used in the current study.