Karyotypes and chromosomal characteristics of both minor and major rDNAs in four fish species known popularly as “lambaris”, namely Astyanax
abramis (Jenyns, 1842), Astyanax
asuncionensis Géry, 1972, Astyanax
correntinus (Holmberg, 1891) and Astyanax sp. collected from downstream of the Iguassu Falls (Middle Paraná River basin), preservation area of the Iguassu National Park, were analyzed by conventional and molecular protocols. Astyanax
abramis had diploid chromosome number 2n=50 (4m+30sm+8st+8a) and single AgNORs (pair 22), Astyanax
asuncionensis had 2n=50 (8m+24sm+6st+12a) and single AgNORs (pair 20), Astyanax sp. had 2n=50 (4m+26sm+8st+12a) and single AgNORs (pair 25), and Astyanax
correntinus had 2n=36 (12m+16sm+2st+6a) and multiple AgNORs (pairs 12, 15, 16, 17). FISH with 18S rDNA showed a single site for Astyanax
abramis, Astyanax
asuncionensis and Astyanax sp. and multiple for Astyanax
correntinus (14 sites). FISH with 5S rDNA showed single 5S-bearing loci chromosome pair only for Astyanax
asuncionensis and multiple for Astyanax
abramis (four sites), Astyanax
correntinus (five sites) and Astyanax sp. (four sites). Distinct distribution patterns of heterochromatin were observed for karyotypes of all species, with the exception of the first acrocentric chromosome pair characterized by centromeric, interstitial-proximal and telomeric blocks of heterochromatin on the long arm, which may represent homeology between karyotypes of Astyanax
abramis and Astyanax
asuncionensis. Our study showed species-specific characteristics which can serve in diagnosis and differentiation between Astyanax
abramis and Astyanax
asuncionensis, considered cryptic species, as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically. In addition, the data obtained from first cytogenetic studies in Astyanax
correntinus suggest a high similarity with Astyanax
schubarti Britski, 1964, suggesting that these species may belong to the same morphological group and that can be phylogenetically related.