Abstract. Anthropogenic activities have significantly altered freshwater fish communities. Extirpations of deepwater coregonines (Coregonus spp.), a diverse group of fish species, have left vast areas of the Laurentian Great Lakes devoid of a deepwater fish community. Currently, fisheries managers are considering restoring populations by reintroducing deepwater coregonines from Lake Superior and Lake Nipigon. However, little is known about the historical ecology of deepwater coregonines, and species characterization has proved difficult. We used stable isotope analysis of museum-preserved and contemporary specimens to investigate if (1) coregonine species historically occupied distinct niches and (2) the pattern of trophic niche partitioning has changed over the last century. Across all lakes, individual species occupied distinct trophic niches, confirming that these species were ecologically distinct. Understanding trophic niche partitioning helps resolve uncertainty about distinctness of species within and across lakes and may provide a better ecological basis for rehabilitation of Great Lakes food webs and ecosystems