Brook trout (Salvelinus fontinalis) often exist as highly differentiated populations, even at small spatial scales, due either to natural or anthropogenic sources of isolation and low rates of dispersal. In this study, we used molecular approaches to describe the unique population structure of brook trout inhabiting the Shavers Fork watershed, located in eastern West Virginia, and contrast it to nearby populations in tributaries of the upper Greenbrier River and North Fork South Branch Potomac Rivers. Bayesian and maximum likelihood clustering methods identified minimal population structuring among 14 collections of brook trout from throughout the mainstem and tributaries of Shavers Fork, highlighting the role of the cold-water mainstem for connectivity and high rates of effective migration among tributaries. In contrast, the Potomac and Greenbrier River collections displayed distinct levels of population differentiation among tributaries, presumably resulting from tributary isolation by warm-water mainstems. Our results highlight the importance of protecting and restoring cold-water mainstem habitats as part of region-wide brook trout conservation efforts. In addition, our results from Shavers Fork provide a contrast to previous genetic studies that characterize Appalachian brook trout as fragmented isolates rather than well-mixed populations. Additional study is needed to determine whether the existence of brook trout as genetically similar populations among tributaries is truly unique and whether connectivity among brook trout populations can potentially be restored within other central Appalachian watersheds.