<p>There is limited understanding of the genetic status and structure of Sauger (<i>Sander canadensis</i>) populations in Manitoba, Canada. We used twelve microsatellite loci to resolve genetic diversity and structure of sauger (<i>n</i> = 872) across nine waterbodies in the Nelson-Churchill watershed. Five waterbodies contained low numbers of walleye × sauger hybrids. Overall, genetic diversity were highest in Lake Winnipeg, the largest lake in the watershed, and lowest in Lakes Manitoba and Winnipegosis. Despite their close geographic proximity, genetic differentiation between Lake Winnipeg and Lake Manitoba/Lake Winnipegosis were moderately high and were found to have low levels of historical and on-going gene flow. On the other hand, high connectivity, based on low levels of genetic differentiation and high estimates of on-going gene flow were found between Lake Winnipeg and the Red River, Assiniboine River, Lac du Bonnet, and Stephens Lake, which span a larger geographic area but are more connected in the watershed. Clustering and genetic differentiation-based methods indicated that fish in Lake Winnipeg and Lake Manitoba/Lake Winnipegosis belong to different genetic groups, but most individuals from all sites exhibited some level of ancestral admixture, particularly fish sampled in the river systems. This suggests that populations in Lake Winnipeg and Lake Manitoba/Winnipegosis should be managed separately, and indicates an important role for the maintenance of northern and southern river populations as reservoirs of genetic diversity. The results of this study will assist in defining management units based on genetic structure and selecting source sauger populations for restoration programs.</p>