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In the North American longleaf pine (Pinus palustris) ecosystem, the Gopher Tortoise (Gopherus polyphemus) is a keystone species that has declined significantly over the last century. Habitat degradation and fragmentation may have caused G. polyphemus to become separated into small, isolated local populations that suffer from decreased genetic diversity or inbreeding depression. Here we use genome-scale methods to sequence thousands of loci for 336 G. polyphemus individuals from 11 sites across southern Alabama to estimate population genetic structure and levels of genetic diversity. We found a pattern of isolation by distance among samples, where geographic distance predicted genetic difference. Principal components and structure analyses supported the existence of three weak genetic populations comprising individuals from (1) Fred T. Stimpson State Game Sanctuary and Perdido Wildlife Management Area, (2) Conecuh National Forest and Solon Dixon Forestry Education Center, and (3) Geneva State Forest Wildlife Management Area. We did not observe strong variation in genetic diversity or effective population size metrics among sampling locations or genetic populations identified by population structure analyses. Our results suggest that G. polyphemus historically operated on larger geographic scales than those considered by contemporary mark-recapture studies. Absence of variation in population genetic metrics suggests that either effects of fragmentation have not manifested themselves, or that the effects are similar across all locations. Given the common use of translocations in Gopher Tortoise management, we provide a framework for tortoise translocations based on our genomic data. [ABSTRACT FROM AUTHOR]