Gopherus polyphemus has experienced severe population declines, especially in the western portion of its range. As a consequence, G. polyphemus may have experienced population bottlenecks that resulted in a decrease in genetic diversity and an accumulation of deleterious alleles. The importance of genetic diversity has been well-documented for several fitness parameters (e.g., survival, disease resistance, growth and developmental rates, and developmental instability). Western populations of G. polyphemus in south Mississippi (USA) have lower hatching success (16.7–48%) than that found in eastern populations (67–97%). Even under laboratory conditions, approximately 40% of the eggs still failed to hatch, suggesting that intrinsic (egg quality) factors may be affecting development. Using nine microsatellite loci, we genotyped individuals from four populations in south Mississippi and one eastern population (Fort Benning, GA) and compared several genetic diversity indices (e.g., allelic richness, expected heterozygosity, and percent polymorphic loci) with published data from populations in the eastern portion of the range, such as populations east of the Mobile and Tombigbee rivers. We found significantly lower genetic diversity in the four Mississippi populations than in the eastern populations. However, these findings only demonstrate that these populations have low genetic diversity, and establishing any causal relationship between low genetic diversity (or other intrinsic factors; e.g., female condition) and reduced reproductive success should be further investigated.