Identifying units of conservation of aquatic species is fundamental to informed natural resources science and management. We used a combination of mitochondrial and nuclear molecular methods to identify potential units of conservation of Westslope Cutthroat Trout Oncorhynchus clarkii lewisi, a taxon native to montane river basins of the northwestern United States and southwestern Canada. Mitogenomic sequencing identified two major lineages composed of nine monophyletic clades, and a well-supported subclade within one of these, largely delineated by river basins. Analyses of microsatellites and single nucleotide polymorphisms corroborated most of these groupings, sometimes with less resolution but demonstrating more complex connections among clades. The mitochondrial and nuclear analyses revealed that Pleistocene glacial cycles profoundly influenced the distribution and divergence of Westslope Cutthroat Trout, that this taxon crossed the Continental Divide in two separate events, and that genetically pure but nonindigenous fish were widely distributed. Herein, we recognize nine geographically discrete, cytonuclear lineages largely circumscribed by major river basins as potential units of conservation: (1) John Day; (2) Coeur d’Alene; (3) St. Joe; (4) North Fork Clearwater; (5) Salmon; (6) Clearwater headwaters; (7) Clearwater-eastern Cascades; (8) neoboreal, consisting of most of the Columbia upstream from central Washington,the Fraser in British Columbia, and the South Saskatchewan in Alberta; and (9) Missouri.