Cyphocharax magdalenae, a Colombian freshwater fish species, plays a vital role in nutrients distribution and serves as a significant food source for other fish species and local fishing communities. Considered a short-distance migratory species, C. magdalenae populations face substantial extinction risk due to human activities impacting their habitats. To address the lack of knowledge on genetic diversity and population structure, this study used next-generation sequencing technology to develop species-specific microsatellite loci and conducted a population genetics analysis of C. magdalenae in the middle and lower sections of the Cauca River, Colombia. Out of 30 pairs of microsatellite primers evaluated in 324 individuals, 14 loci were found to be polymorphic, at linkage equilibrium and, in at least one population, their genotypic frequencies were in Hardy-Weinberg equilibrium. Results showed high genetic diversity levels compared to other neotropical Characiformes, with inbreeding coefficients similar to those reported for phylogenetically related species. Moreover, C. magdalenae exhibits seasonal population structure (rainy-dry) consisting of two genetic stocks showing bottleneck signals and high effective population sizes. This information is essential for understanding the current species genetics and developing future management programs for this fishery resource.