As anthropogenic impacts accelerate changes to landscapes across the globe, understanding how genetic population structure is influenced by habitat features and dispersal is key to preserving evolutionary potential at the species level. Furthermore, knowledge of these interactions is essential to identifying potential constraints on local adaptation and for the development of effective management strategies. We examined these issues in Brook Trout (Salvelinus fontinalis) populations residing in the Upper Hudson River watershed of New York State by investigating the spatial genetic structure of over 350 fish collected from 14 different sampling locations encompassing three river systems. Population genetic analyses of microsatellite data suggest that fish in the area exhibit varying degrees of introgression from nearby State-directed supplementation activities. Levels of introgression in these populations correlate with water-way distance to stocking sites, although genetic population structure at the level of individual tributaries as well as their larger, parent river systems is also detectable and is dictated by migration and influenced by habitat connectivity. These findings represent a significant contribution to the current literature surrounding Brook Trout migration and dispersal, especially as it relates to larger interconnected systems. This work also suggests that stocking activities may have far-reaching consequences that are not directly limited to the immediate area where stocking occurs. The framework and data presented here may aid in the development of other local aquatic species-focused conservation plans that incorporate molecular tools to answer complex questions regarding diversity mapping, and genetically important conservation units.