Fatty acid signatures (FAS) are currently used in food web studies to provide insights into long term feeding habits of predators based on the degree of similarity between their FAS and that of their prey. To date, FAS data of fish from Lake Ontario are limited and are required to better understand possible energy connections between nearshore and offshore environments. In this study, three major prey fish (alewife - Alosa pseudoharengus, rainbow smelt - Osmerus mordax, and round goby - Neogobius melanostomus) were collected at three sites along the south shore of Lake Ontario (Olcott, Rochester, Oswego) at nearshore (m) and offshore (>70 m) locations. Using multivariate statistics, we will compare species FAS as well as their spatial variation. These data will further our understanding of predator-prey interactions in Lake Ontario’s food web.