Abstract In a system that uses supplemental stocking to enhance a fishery that serves a dual purpose, an understanding of the contributions from natural and hatchery-produced fish is important so that hatchery resources can be appropriately allocated. Kokanee Oncorhynchus nerka were first stocked in Flaming Gorge Reservoir (FGR), Wyoming–Utah, in 1963 and serve a dual purpose as a prey resource and sport fish. Although natural recruitment occurs in the reservoir, a supplemental stocking program was initiated in 1991. We sought to identify the natal origin (i.e., natural, hatchery) of kokanee in FGR using otolith microchemistry. We evaluated return to the creel, composition of spawning aggregates, and growth of kokanee in FGR and focused on differences associated with natal origin. We analyzed kokanee otoliths that we collected from hatcheries (n = 60) and FGR (n = 1,003) for the strontium isotope ratio, 87Sr/86Sr, using laser ablation and a multicollector inductively coupled plasma mass spectrometer. We conducted Kruskal–Wallis tests to compare the strontium isotope ratios from the otolith edge of kokanee that we sampled from hatcheries and FGR. Based on 87Sr/86Sr ratios, we could distinguish natural-origin kokanee from 11 of the 12 hatcheries (P < 0.01); however, the Wigwam Hatchery was not significantly different from FGR (P = 0.84). We used model-based discriminant function analysis to assign natal origins for kokanee caught in FGR. Hatchery contribution to the population at large varied from 21 to 50% among year classes from 2014 to 2018. The percentage of hatchery origin kokanee in the creel (18–50%) was similar to what we observed in the population. Hatchery-produced kokanee contributed a higher proportion to tributary-spawning aggregates (40–90%) than shoreline-spawning aggregates (19–58%) by sample year. Growth of natural and hatchery kokanee was similar, suggesting similar performance in the system. Results from this study identify that hatchery supplementation contributes to the population and recreational harvest of kokanee in FGR. This research also provides insight into the ecology of kokanee that is useful for better understanding kokanee population dynamics in reservoir systems.