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. A. abramis had diploid chromosome number 2n=50 (4m+30sm+8st+8a) and single AgNORs (pair 22), A. 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 A. 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 A. abramis, A. asuncionensis and Astyanax sp. and multiple for A. correntinus (14 sites). FISH with 5S rDNA showed single 5S-bearing loci chromosome pair only for A. asuncionensis and multiple for A. abramis (four sites), A. 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 A. abramis and A. asuncionensis. Our study showed species-specific characteristics which can serve in diagnosis and differentiation between A. abramis and A. 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 A. correntinus suggest a high similarity with A. schubarti Britski, 1964, suggesting that these species may belong to the same morphological group and that can be phylogenetically related.