The forage fish communities of the Laurentian Great Lakes continue to experience changes that have altered ecosystem structure, yet little is known about how they partition resources. Seasonal, spatial and body size variation in δ13C and δ15N was used to assess isotopic niche overlap and resource and habitat partitioning among the five common offshore Lake Ontario forage fish species (n = 2037) [Alewife (Alosa pseudoharengus), Rainbow Smelt (Osmerus mordax), Round Goby (Neogobius melanostomus), and Deepwater (Myoxocephalus thompsonii) and Slimy (Cottus cognatus) Sculpin]. Round Goby had the largest isotopic niche (6.1‰2, standard ellipse area (SEAC)), followed by Alewife (3.4‰2) while Rainbow Smelt, Slimy Sculpin and Deepwater Sculpin had the smallest and similar niche size (1.7-1.8‰2), with only the Sculpin species showing significant isotopic niche overlap (>63%). Stable isotopes in Alewife, Round Goby and Rainbow Smelt varied with location, season and size, but did not in the Sculpin spp. Lake Ontario forage fish species have partitioned food and habitat resources, and non-native Alewife and Round Goby have the largest isotopic niche, suggestive of a boarder ecological niche, and may contribute to their current high abundance.