Salmonids are ecologically and economically important fishes in Lake Ontario but declines in Alewife (Alosa pseudoharengus), recently the primary prey item, may cause shifts in salmonid diets. Improving our understanding of salmonid trophic niches and diets and whether these have changed over time will provide insight into salmonid foraging patterns, ecology, and management. Using three stable isotopes (δ13C, δ15N and δ34S) and Bayesian models, we estimated core trophic niches, niche overlaps among species, and diets for six salmonid species from Lake Ontario sampled during 2018. We used fin and muscle tissue with different temporal assimilation periods to assess seasonal variability in diet, and investigated salmonid trophic niches and diets among three major spatial units (East, Central, West). Overall, core trophic niches overlapped across all species, except Lake Trout (Salvelinus namaycush). Consistent with previous lake-wide analyses of Lake Ontario salmonid diets, Alewife were the preferred prey fish, although Lake Trout and Atlantic Salmon (Salmo salar) had more varied diets, particularly based on white muscle. Fin-based isotope mixing models predicted greater consumption of shallow (< 30 m depth) Round Goby (Neogobius melanostomus) for Lake Trout, Brown Trout (Salmo trutta), and Rainbow Trout (Oncorhynchus mykiss) than other salmonids, indicating seasonal variation in diets. Lake Trout and Brown Trout also varied across spatial units with greater Round Goby consumption in western Lake Ontario. Spatially distinct diets suggest that Brown Trout and Lake Trout populations should be monitored regionally. Understanding prey consumption trends are important to effectively manage fish populations, especially considering on-going ecological changes in the Great Lakes.