Microsternarchus bilineatus is a neotropical electric fish species widely distributed in Amazonian ecosystems. This work reports the first karyotypic description of the species M. bilineatus, which presented 2n = 48 chromosomes, with a distinct karyotypic formula between the sexes: males with 21 metacentric (m)/submetacentric (sm) + 27 subtelocentric (st)/acrocentric (a) and fundamental number (FN) = 69 and females with 20 m/sm + 28 st/a and FN = 68. We found a probable recent sex system of XX/XY type. The nucleoli organizer regions (NORs) were multiple terminally located, and the heterochromatic blocks were mostly pericentromeric. The 18S rDNA markings confirmed NORs and their distinction between sexes, which suggested some differential role of this gene related to gender in this species. The 5S rDNA presented terminal markings on a single chromosome pair, with no distinction between sexes, and the telomeric probes have shown a uniform pattern in males and females.
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