Lake (Coregonus clupeaformis) and round (Prosopium cylindraceum) whitefish are sympatric benthivores in Lake Huron that are thought to coexist via niche partitioning. However, little is known about long-term resource use and niche overlap across different temporal scales. We used a multiyear (2010–2012) and multi-tissue (liver, muscle, and bone layers) isotopic niche analysis to characterize and compare resource use by lake and round whitefish across several time scales. Lake whitefish consistently used more diverse, 13C-depleted (mean δ13C = −21.9‰) and 15N-enriched (mean δ15N = +9.3‰) resources than round whitefish (mean δ13C = −18.2‰; mean δ15N = +8.3‰). Niche overlap occurred only in liver, representing the spawning period, while niche segregation was highest in juvenile life stages. Individuals of both species made variable resource shifts among time periods, suggesting that spawning aggregations are composed of individuals representing a variety of feeding strategies and locations. Our study confirms that differential resource use is an important strategy for these fish as adults and demonstrates life-long niche partitioning beginning before age-2.