The monotypic Astroblepidae fish family includes 81 species distributed along Central and Northern Andes in South America and Panamá in Central America; most aspects regarding its biology, taxonomy, and chromosomal features remain largely unknown. This study reports the karyotype of two sympatric Astroblepus species from the Colombian Andes, aiming to provide novel information on karyotype organization and reveal possible chromosomal rearrangements occurred on these species, through mapping of different repetitive DNA classes, including microsatellites and ribosomal DNA multigene families. The results showed differences in the chromosome number and karyotypic formula: Astroblepus grixalvii had 2n = 52 (28m+8sm +12st +4a) with the metacentric pair number 1 as the largest chromosome pair, whereas Astroblepus homodon had 2n = 54 (30m+8sm +8st +8a) and less evident chromosome size differences. Microsatellite probes marked the tips of all chromosomes in both species except the short arms of acrocentric pair numbers 24 and 25 in A. homodon. Each ribosomal probe marked different chromosome pairs in both species. Microsatellite patterns suggest that the 2n increase probably involved a centric fission event that occurred during the evolutionary history of these species. This is the first karyotype description of an Astroblepus species and it contributes to the theoretical framework about the karyoevolutionary trends within Loricarioidei.