Astyanax has been the subject of extensive cytogenetic studies due to its wide karyotypic diversity. This genus comprises species complexes, namely groups of fish of difficult morphological differentiation, such as the bimaculatus complex, which includes the characids with a rounded humeral spot. Thence, the present study proposed to accomplish a cytogenetic characterization of two species of this complex: A. asuncionensis and A. altiparanae, aiming to find chromosomal markers that differentiate these species, as well as achieve a better understanding of the karyotype evolution in the genus. For this we used different techniques of chromosome banding as C-banding, impregnation by silver nitrate, fluorochrome staining and FISH with 18S rDNA probe. This is the first cytogenetic study in A. asuncionensis, from Miranda river, which presented 2n = 50 and 18 m + 22sm + 6st + 4a (FN = 96) and single NORs. The populations of A. altiparanae also presented 2n = 50, but with different karyotypic formulae: the population of the Quexada river presented 16 m + 24sm + 4st + 6a (FN = 94) and the Esperança stream and Jacutinga river showed 16 m + 20sm + 4st + 10a (FN = 90). All analyzed populations showed an interindividual variation in the number and location of the nucleolar organizer regions (NORs). Single and multiple NORs were detected either by impregnation with silver nitrate or by FISH with 18S rDNA probe. After C-banding, the two species differed in relation to the composition and heterochromatin distribution. The meiotic cells of A. altiparanae male individuals were also analyzed, showing that, despite the high karyotype variability, chromosome pairing occurs normally. The data show that A. altiparanae and A. asuncionensis share some characteristics with other species of the bimaculatus complex, suggesting a close phylogenetic relationship among those species. However, some features can be used as differentiation chromosomal markers in altiparanae/asuncionensis morphotypes, which could result from a natural speciation process.