Comprising a large number of species, the genus Astyanax has been intensively studied by several approaches to elucidate its evolutionary relationships. Such studies have demonstrated that many nominal species are artificial clusters where distinct taxa are grouped under the same denomination. Astyanax aff. fasciatus stands out due to its high karyotypic diversity, since cytogenetic studies have reported three standard cytotypes (2n = 46, 48, and 50), as well as cases of sympatry between cytotypes, variant cytotypes, and B chromosomes. In this study, we attempted to evaluate the reliability of the chromosomal differences in relation to the analysis of the ATPase6/8 mitochondrial DNA (mtDNA) sequence, thereby providing subsidies to the evolutionary reconstruction of this group. Nine populations from four distinct hydrographic basins along Southeastern Brazil were analyzed. These are the first cytogenetic data collected for four of them. Fluorescent in situ hybridization with 5S rDNA probe evidenced the presence of a standard phenotype for the group and the existence of a new arrangement in the individuals from Ribeira de Iguape River. Besides the karyotypic variation, the genetic distance was low among the studied populations and some aspects of the evolutionary relationships among distinct cytotypes/populations could be ascertained by phylogeographic studies. The incipient molecular structuring of certain cytotypes in different hydrographic basins indicates the role of different evolutionary processes on the diversification of the group.