Populations of the endangered giant kangaroo rat (Dipodomys ingens) have decreased because of habitat fragmentation and isolation over the past 100 y. Changes in population structure due to habitat fragmentation can significantly affect the population size and the dispersal of these animals compromising long-term sustainability of each fragmented population. We collected small ear clippings from male and female giant kangaroo rats from six sites in the southern San Joaquin Valley to determine the genetic population structure of this species in this part of their range. Multilocus F-statistics indicated that the six populations of giant kangaroo rat are not composed of randomly mating populations and that genetic drift and inbreeding are major determinants of population structure. Furthermore, F-statistics confirmed a significant decrease in observed heterozygosity. Genetic distance analyses did not support the hypothesis that geographically distant populations would exhibit greater genetic differentiation. We also compared our data to published estimates of genetic diversity of giant kangaroo rats in populations to the west and north, the other large population centers of this species.