The Texas kangaroo rat (Dipodomys elator) is listed as a threatened species in Texas because of its scarcity and small geographic range. We assessed patterns of genetic diversity in D. elator that could affect extinction risk or influence management decisions. Specific objectives included: 1) document levels of genetic diversity, 2) document the degree and patterns of genetic divergence among localities, and 3) compare levels of genetic diversity between different time periods at the same locality. Portions of the mitochondrial genome (mtDNA; control region, cytochrome c oxidase subunit I, and cytochrome b) were sequenced and nuclear microsatellites were examined. Low mtDNA diversity was observed, which could be explained by an historical, species-wide genetic bottleneck. In contrast, microsatellites exhibited ample variation, and analyses were conducted using data from 11 loci and four populations (designated Quanah, Iowa Park, Vernon, and Harrold). Allelic diversity and heterozygosity were similar between populations and temporal samples. Estimates of effective population size (Ne) ranged from 5 to 856, depending on method and population, with Iowa Park showing consistently lower values than Quanah. All methods addressing population structure indicated that the Iowa Park population was divergent from the others, with Vernon and Harrold showing a somewhat intermediate relationship but with a closer affiliation with Quanah than Iowa Park, despite their closer proximity to Iowa Park. This pattern did not conform to isolation by distance, thus genetic drift appears to have played a greater role than gene flow in establishing genetic structure. There was much less difference between temporal samples compared to geographic samples, indicating that genetic drift has had only minimal impacts in shifting allelic frequencies over the time periods examined (17–36 years).