Conservation and management issues related to genetic diversity and stock structure of native populations of muskellunge (Esox masquinongy) have largely been unexplored. In Lake Huron’s North Channel and Georgian Bay, Muskellunge populations have been impacted by historical commercial fisheries, recreational fisheries, loss of spawning habitat, historical water quality issues, and ecosystem changes. To determine the spatial genetic structure of native Muskellunge in Lake Huron and Georgian Bay, spawning adults were sampled from 10 sites in the North Channel and eastern Georgian Bay. Genotyping with 20 microsatellite DNA loci showed substantial spatial genetic structure, with significant pairwise divergences among spawning sites. Individual- and population-based analyses revealed hierarchical population structuring, with strong patterns of spawning site fidelity and isolation by distance; very low levels of dispersal and gene flow over historical and contemporary timescales were indicated. Estimation of effective population sizes highlighted the limited genetic resources that are present in these localized populations. The present results show that Muskellunge in Lake Huron and Georgian Bay consist of multiple small populations with limited ranges and high site fidelity and should be managed accordingly.