In fisheries management, understanding anthropogenic impacts on fish population genetic structure is essential because genetic diversity is a fundamental attribute contributing to a species’ evolutionary capacity. An extended history of supplemental stocking has led to the introgression of genes from non-local, hatchery-reared brook trout (Salvalinus fontinalis) into natural Adirondack populations in the state of New York. Managers have therefore gone to great lengths to protect known or suspected pristine “heritage” populations, but the genetic integrity of most populations is unknown. We used 11 microsatellite loci to examine a putative, but as yet unconfirmed “heritage” population in Dix Pond (Essex County, New York), in an effort to confirm its genetic uniqueness, quantify genetic diversity, and determine the geographic extent of the population. No spatial population structure was found within the Dix Pond/Elk Lake watershed, with minimal signs of introgression from historical stocking. The Dix/Elk population showed allelic richness, and effective population size comparable to the highest diversity heritage population among the four that we used for comparison. These patterns support continued heritage status for the Dix Pond population and recognition of the entire Dix-Elk watershed as habitat for this strain. We conclude this study by discussing how the genetic techniques employed here may help to inform future management decisions associated with the conservation and protection of imperiled populations throughout the globe.