From a conservation management perspective it is important to understand how genetic diversity is partitioned across a species’ range, including (1) identification of evolutionarily distinct units versus those recently isolated through anthropogenic activities and (2) the relative genetic contributions among components of fragmented (meta)populations. To address these questions, we investigated the phylogeography and metapopulation structure among relict populations of the endangered giant kangaroo rat (Dipodomys ingens) in the highly altered San Joaquin Desert Ecosystem. This keystone species underwent a ~97% range reduction over the past century, resulting in a current range that is highly fragmented, with two dominant northern and southern populations occurring 150 km apart. We sequenced >800 bp of mitochondrial DNA and genotyped 17 nuclear microsatellites in >275 D. ingens to assess the evolutionary relationship of these populations as well as the genetic structure within the northern metapopulation. A Bayesian Skyline Plot indicated that the species experienced a demographic expansion toward the end of the Pleistocene, with a recent population decline. Northern and southern D. ingens split 1,857–13,443 years ago, prior to the massive conversion of the San Joaquin Valley to irrigated agriculture. We recommend that the northern and southern populations of D. ingens be re-classified as distinct population segments under the United States Endangered Species Act. We also observed population structure and asymmetrical migration within northern D. ingens where the Tumey Hills acted as a source contributing gene flow to all peripheral populations. This emphasized the importance of this location in the conservation of the metapopulation as a whole.