We conducted a genetic study of the largest cluster of US federally threatened Gopher Tortoise (Gopherus polyphemus) colonies. Our objectives were to (1) identify genetic variation within and among colonies across the landscape; (2) determine which factors are important in affecting genetic variation, including land use, habitat quality, and population size; and (3) determine whether genetic partitioning among populations exists and how this relates to (a) geographic distance between sites, (b) Gopher Tortoise natural history and spatial ecology, and (c) land-use history. We studied genetic variability of nine microsatellite DNA loci for 340 adult tortoises from 34 colonies separated by 1.3–45.1 km across a 56,000-ha military installation. Overall genetic variation was low across the landscape and within colonies. Observed heterozygosity (Ho) of tortoise colonies was 49% and allelic richness was 52% of that found in populations located in the eastern portion of the species distribution where habitat is naturally more continuous. Our single colony with highest genetic variation had Ho that was 57% and allelic richness that was 60% of eastern colonies. Genetic variation was greatest in sites with suitable habitat. We found weak to no genetic structure across the 45-km landscape (FST = 0.031; DST = 0.006) and evidence for only one genetic group (K). Although landscape reconfiguration to create sites for military activity has redistributed tortoise colonies and home ranges, we concluded that weak population structure is natural across our study area. Comparison to similar results from a cluster of connected eastern colonies suggests this is a general characteristic of tortoises across large, continuous landscapes and that populations are composed of multiple colonies across the landscape and are naturally large in spatial extent. To alleviate the tortoise-human land use conflict on Camp Shelby, Mississippi, USA and to ensure these created areas continue to benefit tortoises in the long term, maintenance of forest habitat surrounding these created open areas is required. We recommend managing tortoises at Camp Shelby as one unit.