In the 1960s, as a part of the construction of the Furnas Hydroelectric Power Dam, Minas Gerais, Brazil, the Rio Piumhi was diverted from the Rio Grande drainage to the São Francisco River basin, with the resulting introduction of species from one basin to the other. Chromosomal characteristics of various populations of Astyanax fasciatus sensu Eigenmann from the transect region were studied using cytogenetic techniques, with the goal to identify and map the dispersal of invasive species in the Rio São Francisco. Populations of the Rio Grande and Rio Piumhi are distinct from those of the São Francisco basin based on chromosome markers associated to the heterochromatin, Ag-NORs, 18S rDNA, 5S rDNA, and As-51 satellite DNA sites. Individuals with karyomorph originally present in the Piumhi drainage are now present in the São Francisco basin, downstream from the transposition channel. This expansion of the distribution corroborates a hypothesis of progressive substitution of native populations by the invasive Piumhi form.