<p>The Timber Rattlesnake (<i>Crotalus horridus</i>) is the primary rattlesnake species inhabiting the temperate deciduous forests of eastern North America and faces conservation challenges across its range. In North Carolina, the Timber Rattlesnake is listed as a Species of Special Concern, with habitat loss and fragmentation from human encroachment as its primary threat. To effectively manage and conserve Timber Rattlesnake populations, land managers must first understand their genetic diversity and population structure. We use 19 microsatellite markers to estimate genetic diversity parameters from 125 individuals sampled opportunistically across a broad geographic area in Western North Carolina. Our findings reveal robust genetic diversity and three management units, which will assist land managers in formulating effective conservation action plans.</p>