Discontinuous genetic structure is widely used to delineate local, regional, and phylogenetic groups within species for conservation and management purposes. We used microsatellite markers to assess the genetic distinctiveness of putative stocks and populations of lake whitefish (Coregonus clupeaformis) in Ontario waters. Analysis of spawning aggregations in eastern Lake Ontario showed fish from Chaumont Bay, New York, to be weakly differentiated from spawning whitefish in and near the Bay of Quinte, Ontario. No significant differences were found between lake- and bay-spawning aggregations within the Bay of Quinte. These same genetic tools were used to test the distinctiveness and evolutionary significance of Lake Simcoe lake whitefish as a designatable unit (DU) under guidelines established by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). Although there was marked differentiation among populations from across Ontario, the Lake Simcoe population was closely allied with lake whitefish populations from Lake Ontario and Lake Huron, suggesting that a distinct status is not warranted on genetic grounds. This work demonstrates how assessing hierarchical diversity under COSEWIC’s framework can provide key information of the status of exploited populations for fishery management.