Astyanax is one of the most abundant and diverse taxa of fishes in the Neotropical region. In order to increase the amount of cytogenetic information for Astyanax as well as to exhibit data to subsidize future taxonomic studies, this work analyzed three species of Astyanax: two species are cryptic, and are here reported to live in syntopy (A. abramis and A. lacustris); the first karyotype description for A. pirapuan is also presented. Cytogenetic analyzes reveal a diploid number of 2n=50 chromosomes for three species, yet with differences in their karyotype morphology. The physical mapping of 18S rDNA showed up to thirteen sites in A. pirapuan and two in A. abramis and A. lacustris. The physical mapping of 5S rDNA has proven to be an effective marker for the characterization of species of Astyanax studied in this work.