Delineating population units is essential for the conservation and management of a species. Applying a genetic approach to delineate units, this study identifies genetic population structure, and landscape resistance to gene flow, of the nationally threatened boreal woodland caribou (Rangifer tarandus caribou) across the ecotypes’ southern range in Saskatchewan. Three genetic clusters were delineated across the study area, with moderate genetic connectivity identified with Manitoba. Isolation-by-distance was found to be significant across Saskatchewan, and within each genetic cluster. Gene flow across clusters in Saskatchewan was high (FST = ~0.01), with genetic connectivity being lowest for the south-central cluster surrounding Prince Albert National Park (FST = ~0.03). Resistance to gene flow was identified with the following landscape variables: water, forestry, roads, wildfire, and low suitability habitat. Careful consideration of these variables in range planning will help to maintain genetic connectivity of boreal caribou across its southern range in Saskatchewan.