Anostomidae are a neotropical fish family rich in number of species. Cytogenetically, they show a conserved karyotype with 2n = 54 chromosomes, although they present intraspecific/interspecific variations in the number and chromosomal location of repetitive DNA sequences. The aim of the present study was to perform a comparative description of the karyotypes of two populations of Leporinus friderici Bloch, 1794 and three populations of Leporellus vittatus Valenciennes, 1850. We used conventional cytogenetic techniques allied to fluorescence in situ hybridization, using 18S ribosomal DNA (rDNA) and 5S rDNA, a general telomere sequence for vertebrates (TTAGGG)n and retrotransposon (RTE) Rex1 probes. The anostomids in all studied populations presented 2n = 54 chromosomes, with a chromosome formula of 32m + 22sm for L. friderici and 28m + 26sm for L. vittatus. Variations in the number and location of the 5S and 18S rDNA chromosomal sites were observed between L. friderici and L. vittatus populations and species. Accumulation of Rex1 was observed in the terminal region of most chromosomes in all populations, and telomere sequences were located just on all ends of the 54 chromosomes in all populations. The intraspecific and intergeneric chromosomal changes occurred in karyotype differentiation, indicating that minor chromosomal rearrangements had present in anostomid species diversification.