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. These issues were addressed for Brook Trout (Salvelinus fontinalis) populations residing in the Upper Hudson River watershed of New York State by investigating the spatial genetic structure of over 300 fish collected from 14 different sampling locations encompassing three river systems (the Upper Hudson, the Boreas, and the Schroon). The results of this work suggest that fish in the area (i) exhibit varying degrees of introgression from State-directed stocking activities (ii) exhibit genetic population structure at the level of individual tributaries as well as the larger river systems where they are found, dictated by migration and influenced by habitat connectivity, and (iii) demonstrate comparatively similar measures of genetic diversity but varied measures of effective population size based on sampling location. These findings represent a significant contribution to the current literature surrounding Brook Trout migration and dispersal, especially as it relates to larger interconnected systems. Finally, this study is concluded with a discussion about how the methods used here may aid in the development of other species-focused conservation plans that incorporate an evolutionary perspective.