In the southeastern United States, habitat loss has fragmented the landscape and isolated many populations of this region's flora and fauna, which has presumably resulted in smaller population sizes and reduced levels of genetic diversity. For example, forestry practices and anthropogenic disturbances are both cited as factors fragmenting the once extensive range of Gopherus polyphemus. One localized, but extreme, source of fragmentation was the impoundment of the Chattahoochee River in 1963 to form Walter F. George Reservoir along the border of Georgia and Alabama. The formation of this reservoir isolated populations of G. polyphemus on two newly created islands providing a natural laboratory to explore the demographics and genetic effects of fragmentation on a long-lived species. These populations were first surveyed in 1984 and, 21 years later, we revisited them to collect demographic data and tissue samples for genetic analysis. We genotyped all individuals for 10 microsatellite loci, and we tested these data for bottlenecks and compared them to levels of genetic diversity for populations from other portions of the range. We found 45 and two individuals on the larger and smaller islands, respectively. On the large island, however, the population size was identical to the 1984 survey. Only the population structure based on estimated age differed between the 1984 and 2004 surveys, while population size structure based on carapace length, sex ratio, and sex-specific growth rates did not differ. The population of the large island showed genetic evidence of a past bottleneck. The genetic diversity indices from the population of the large island, however, were comparable to or greater than those found at mainland sites, in particular from western populations.