Muskellunge (Esox masquinongy) are important apex predators that support numerous recreational fisheries throughout the Great Lakes region. Declines in muskellunge abundance from historical overharvest and environmental degradation have threatened the viability of many populations and prompted significant restoration efforts that often include stocking. The goal of our study was to investigate contemporary population structure and genetic diversity in 42 populations of muskellunge sampled across the Great Lakes region to inform future management and supplementation practices. We genotyped 1896 muskellunge (N = 10–123/population) at 13 microsatellite loci. The greatest genetic variation was between populations of Great Lakes origin and populations of Northern (inland) origin, with both groups also exhibiting significant substructure (overall FST = 0.23). Genetic structure was generally correlated with geography; however, we only found marginal evidence of isolation by distance, likely due to high genetic differentiation among proximate populations. Measures of genetic diversity were moderate across most populations, but some populations displayed low diversity consistent with small population sizes or historical bottlenecks. Many of the populations studied displayed evidence of historic introductions and supplemental stocking, including the presence of individuals with primarily non-native ancestry as well as interlineage hybrids. Our results suggest that the historic population structure of muskellunge is largely intact across the Great Lakes region, but also that stocking practices have altered this structure to some degree. We suggest that future supplementation practices use local sources where possible, and incorporate genetic tools including broodstock screening to ensure that non-native muskellunge are not used to supplement wild populations.