Astyanax is a species-rich polyphyletic genus distributed between the southern United States and central Argentina. The genus contains groups of cryptic species, which are difficult to distinguish, and are sometimes identified wrongly. Basic and molecular cytogenetic analyses were run on Astyanax abramis and three junior synonyms of Astyanax lacustris: Astyanax altiparanae, from the upper Paraná River basin, Astyanax asuncionensis, from the lower Paraná basin, and Astyanax jacuhiensis, from the upper Uruguay River. These species all belong to the Astyanax bimaculatus group. All species presented 2n = 50 chromosomes and single nucleolar organizing regions (NORs). In A. altiparanae, the karyotype was 6m + 28sm+4st+12a and the NORs were present in pair 20, while A. jacuhiensis was 8m + 28sm+6st+8a, with NORs in pair 22, and A. asuncionensis was 8m + 24sm+6st+12a, with NORs in pair 20. A. abramis was 4m + 30sm+8st+8a with NORs in pair 22. Fluorescence in situ hybridization revealed single 5S rDNA cistrons in A. altiparanae and A. asuncionensis, and multiple (4) cistrons in A. abramis and A. jacuhiensis. Heterochromatin had a distinct distribution in each species, but was predominantly centromeric and interstitial proximal. In A. abramis and A. asuncionensis, the first acrocentric chromosome pair presented centromeric, telomeric, and interstitial-proximal heterochromatin in the long arm, which may represent the presence of homologous chromosomes in these species. While there are some cytogenetic similarities, differences in the location of 5S rDNA, distribution of heterochromatin, and karyotype formulae contribute to the differentiation of the study species, and support the identification of phylogenetically proximate groups in the "Astyanax clade."