Introduction: The freshwater fish Brycon henni (Characiformes: Bryconidae) is endemic to Colombia and currently considered as “least concern” according to the International Union for Conservation of Nature (IUCN). Objective: To develop microsatellite markers to examine population genetics in Brycon henni. Methods: Using a low-coverage sequenced genomic library, this study developed the first set of microsatellite loci to study the population genetics of this Neotropical species. These loci were used to evaluate the genetic diversity and structure of B. henni from three sites of the Magdalena-Cauca Basin (Colombia). Results: A set of twenty-one polymorphic microsatellite loci was highly informative and revealed that B. henni is evenly genetically structured between two tributaries of the Cauca River separated by only 30 km, a finding that indicates it conforms to reproductively isolated groups. Conclusions: We reported a set of twenty-one polymorphic microsatellite loci that was highly informative and allowed the detection of genetic structure at local and regional scales. This genetic population structure, concordant with that found in eight congeners, is relevant to estimate the B. henni risk categorization as well as for management, conservation, and restocking programs.