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Ancistrus Kner, 1854 is a diverse catfish genus, currently comprising 66 valid species, but karyotype data were recorded for 33 species, although only ten have their taxonomic status defined. Considerable karyotype diversity has been found within this genus, with 2n varying from 34 to 54 and structural variability including heteromorphic sex chromosomes. In many cases, uncertainty on the taxonomic status of the study populations hampers reliable interpretation of the complex chromosomal evolutionary history of the group. This study aims to present the first karyotype data for a population of the Ancistrus sp. collected in Criminoso stream (tributary of the Paraguay River Basin, Mato Grosso do Sul, Brazil) in which a combination of different chromosomal markers was used and results integrated in broad discussion on karyotype evolution in the genus. The specimens presented 2n= 42 with 18m+ 16sm+ 8st and a …