Within many biomes, the cause of phylogeographic structure remains unknown even across regions throughout North America, including within the biodiverse Chihuahuan Desert. For example, little is known about population structure or the diming of diversification of Chihuahuan endemics. In part, this is due largely to the lack of population genomic studies within this region. We generated ultra-conserved element data for the gray-banded kingsnake (Lampropeltis alterna) to investigate lineage divergence and historical demography across the Chihuahuan Desert. We found three unique lineages corresponding to the Trans-Pecos and Mapimian biogeographic regions of the Chihuahuan Desert, and a distinct population in the Sierra Madre Occidental. Using several different mutation rates to calibrate the timing of divergence among these lineages, we demonstrate that lineage divergence likely occurred during the Pleistocene. This also suggests that careful consideration needs to be used when applying mutation rates to ultra-conserved elements. We suggest that biogeographic provinces within the Chihuahuan Desert may have served as allopatric refugia during climatic fluctuations of the Quaternary. This work serves as an important template for further testing biogeographic hypotheses within the region.