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Background: The main cytogenetic studies of the Characidae family comprise the genera Astyanax and Psalidodon involving the use of repetitive DNA probes. However, for the microsatellite classes, studies are still scarce and the function of these sequences in the genome of these individuals is still not understood. Thus, we aimed to analyze and compare the distribution of microsatellite sequences in the species Astyanax bimaculatus and Psalidodon scabripinnis ., Methods: We collected biopsies from the fins of A. bimaculatus and P. scabripinnis to perform cell culture, followed by chromosome extraction, and mapped the distribution of 14 microsatellites by FISH in both species., Results and Discussion: The diploid number observed for both species was 2n = 50, with an acrocentric B microchromosome in A. bimaculatus and a metacentric B chromosome in P. scabripinnis . Regarding FISH, 11 probes hybridized in the karyotype of A. bimaculatus mainly in centromeric regions, and 13 probes hybridized in P. scabripinnis , mainly in telomeric regions, in addition to a large accumulation of microsatellite hybridization on its B chromosome., Conclusion: Comparative FISH mapping of 14 microsatellite motifs revealed different patterns of distribution both in autosomes and supernumerary chromosomes of A. bimaculatus and P. scabripinnis , suggesting independent evolutionary processes in each of these species, representing excellent data on chromosome rearrangements and cytotaxonomy., Competing Interests: The authors declare there are no competing interests., (©2024 de Sousa et al.)