The endangered giant kangaroo rat (Dipodomys ingens, GKR) is a keystone species restricted to the San Joaquin Desert of California, whose range has been reduced to 3% of historical, largely due to habitat loss to irrigated agriculture. The main remaining populations of the GKR are divided into two distinct and fragmented populations 150 km apart: the northern GKR of the Ciervo-Panoche natural area and the southern GKR of the Carrizo Plain. To aid in conservation efforts we sought to understand the demographic history of the species, the historical relationship between the major remaining populations, and to determine how the genetic diversity of the species is partitioned across the range. We set out to achieve this by examining >275 GKR at two mitochondrial DNA regions (>800 bp) and 17 nuclear microsatellites. We estimated that the main populations of GKR split 774-5,600 years ago, prior to agricultural intensification during the 20th century. Within the northern Ciervo-Panoche area we resolved multiple distinct subpopulations, and identified the Tumey Hills as a major source population. Identification of geographically discrete populations of giant kangaroo rats and the relationships among them will aid in conservation decision making by management agencies.