Astyanax bimaculatus, a ubiquitous species in many Neotropical basins, is characterized by a complex taxonomy and are currently considered a species complex. The goal of this study was to analyze 31 populations (N = 136) of this species from southeastern Brazil using cytogenetic techniques: conventional staining, nucleolar organizer region (Ag-NOR), C-banding, and 18S and 5S recombinant DNA fluorescent in situ hybridization (FISH) probes; and molecular techniques: S72, RAG2, and COI. All populations were 2n = 50 (6m + 20sm +18st +6a); Ag-NORs were predominantly simple, C-banding revealed high variation levels within and among basins, and the FISH probes 18S and 5S were restricted to chromosome pairs 14 and 7, respectively. The S72 was uninformative for phylogenetic analyses, and RAG2 showed no variation among populations. The COI gene revealed three haplogroups. The most basal was composed of Pandeiros population (São Francisco Basin) that diverged in the Middle Miocene. The second was composed of A. altiparanae from the Upper Paraná Basin and Espírito Santo Stream (Paraíba do Sul Basin), whereas the third was composed of Astyanax lacustris from São Francisco and coastal basins. The second and third haplogroups diverged in the Pleistocene, indicating that diversification of the bimaculatus complex was driven by tectonic activity and sea-level fluctuations.
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