The genus Astyanax comprises 86 species of fish distributed in Brazilian river basins and is considered of the Incertae sedis group within the family Characidae. This study presents an analysis of 12 specimens of Astyanax jacuhiensis from the Tramandai River Basin, RS Brazil: 6 from the Maquiné River and 6 from the Quadros Lagoon. All specimens showed a diploid number equal to 50 chromosomes with different karyotypic formula between the two localities. The population from the Maquiné River showed 10m+26sm+6st+8a (FN=92). Fish from the Quadros Lagoon showed 12m+20sm+6st+12a (FN=88). AgNORs were evidenced in the short arm of one acrocentric chromosome pair in both populations, confirmed by FISH with the 18S rDNA probe. CMA3 fluorochrome corresponded with the AgNOR sites, while DAPI staining was negative in these regions. C banding revealed that heterochromatin was weakly distributed, mainly in the pericentromeric and terminal regions in most chromosomes. Analyses of male gonadal tissue were conducted with the objective of characterizing the meiotic chromosome behavior in A. jacuhiensis. The following stages were evidenced: spermatogonial with 50 chromosomes, pachytene and metaphase I with 25 bivalents, and metaphase II with 25 chromosomes, thus confirming the diploid number of the species. Chromosomal abnormalities were not observed. This study shows preliminary data on A. jacuhiensis from the Tramandai River Basin, contributing with more chromosomal information for this group of fish.