Maintaining genetic diversity is essential for the long-term conservation of migratory freshwater fishes facing increasing anthropogenic pressures. Prochilodus magdalenae (bocachico), one of Colombia’s most economically and ecologically important freshwater fish species, supports artisanal fisheries, regional food security, and the livelihoods of local communities but has experienced population declines associated with overfishing, habitat degradation, and hydrological alteration. This study evaluated the genetic diversity and population structure of wild and hatchery-associated P. magdalenae from northern Colombia using sequence-based microsatellite genotyping (SSR-GBS). Sixty individuals (48 wild adults from three localities in the Department of Sucre and 12 hatchery-associated fingerlings from Cereté) were genotyped at seven polymorphic microsatellite loci. Wild populations retained high genetic diversity and exhibited weak but significant genetic differentiation (FST = 0.017, p = 0.003), with only 2% of the molecular variation distributed among populations and high estimated gene flow (Nm = 14.899). Hatchery-associated fingerlings consistently exhibited lower allelic richness while maintaining relatively high heterozygosity. Rarefied allelic richness did not differ significantly among populations (Kruskal–Wallis, p = 0.400); however, hatchery-associated fish consistently showed lower values, highlighting the value of allelic richness as a complementary indicator for evaluating hatchery-produced stocks. These findings provide a genetic baseline for the conservation of P. magdalenae and support incorporating routine genetic monitoring into supportive breeding and restocking programs.