Lake Whitefish are a commercially and ecologically valuable freshwater species in Lake Huron. Recent environmental changes have had broad impacts on current populations, which threaten the sustainability of this fishery. To properly conserve this resource, multiple sub-populations in the lake must be adequately represented in management regimes. Of special importance are adult spawning aggregations, which collectively contribute to the productivity of the fishery. However, spawning aggregations in Lake Huron are perceived to represent multiple stocks, adding complexity to fisheries management. The current population structure for adult Lake Whitefish is not well understood at the lake wide scale, and previous assessments have primarily used only evolutionary markers (e.g., genetic and phenotypic markers). A shorter-term, food web based marker would be beneficial for understanding the current population structure of spawning aggregations based on ecological relationships. Stable isotopes are integrated food web tracers that encompass patterns in consumer resource use (i.e., prey, location, habitat). In this study, I used δ13C and δ15N stable isotope analysis to compare prespawning resource use by 32 spawning aggregations spanning most of Lake Huron. I found large isotopic variation across aggregations (ranges: δ13C = 10.2 ‰, δ15N = 5.5 ‰), and spatial heterogeneity in resource use identity and diversity (SEAC = 1.02 – 4.27‰2), indicating that aggregations in different lake areas used more local food webs. I used the isotopic structure of the population to classify individuals into 1 of 4 isotopically distinct clusters. Aggregations were composed of individuals from multiple clusters, indicating highly mixed populations. Aggregations in the Main Basin were particularly mixed (3+ clusters), while those in the North Channel and Georgian Bay represented fewer, and different clusters. To aid interpretation of isotopic structure I investigated factors of location, diet, sampling date, fish sex, and fish size as potential mechanisms of isotopic structuring. Analyses of summer fish and prey baseline variation across lake areas indicate locational feeding patterns are likely the primary mechanism driving ecological structuring. For example, δ13C values of summer assemblages explained 82% of the variation in spawning aggregations at overlapping locations. Lastly, to present an overall view of ecological population structure, I grouped spawning aggregations according to like compositions. To this end, spawning aggregations were divided into 6 putative ecological sub populations that reflect regions of local resource use in the lake. Comparison to the current management scheme suggests that current management units are too small and numerous to reflect the ecology of Lake Whitefish. Stable isotopes provided a powerful approach for identifying ecologically based population structure and composition in an inter-mixed freshwater fishery.