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.
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