Fatty acid profiles are used in food web studies to assess trophic interactions between predator and prey. The present study provides the first comprehensive fatty acid dataset for important prey and predator species in Lake Ontario. Three major prey fish (alewife, rainbow smelt, and round goby) were collected at three sites along the southern shore of Lake Ontario during the spring and fall of 2013, and predator species were collected in similar locations during the summer of 2013. Fatty acid compositions were compared among all prey species, all predator species, and information from both predator and prey was used to infer foraging differences among predators. Seasonal differences in fatty acids were found within each prey species studied. Differences among prey species were greater than any spatio-temporal differences detected within species. Fatty acids of predators revealed species-specific differences that matched known foraging habits. Chinook and Coho salmon, which are known to select alewife as their dominant prey item, had relatively little variation in fatty acid profiles. Conversely, brown trout, lake trout, yellow perch and esocids had highly variable fatty acid profiles and likely highly variable diet compositions. In general, our data suggested three dominant foraging patterns: 1) diet composed of nearly exclusively alewife for Chinook and Coho Salmon; 2) a mixed diet of alewife and round goby for brown and lake trout, and both rock and smallmouth bass; 3) a diet that is likely comprised of forage fishes other than those included in our study for northern pike and chain pickerel.