Two species of horned lizards are sympatric along the periphery of the Salton Trough. Phrynosoma mcallii, endemic to the trough, is of conservation concern because its limited habitat has been fragmented by human activities. A more common and widespread species, Phrynosoma platyrhinos, occurs around the periphery of the trough and much further to the North. The two species are syntopic at a few localities, where morphological intermediates have also been found. Here, we used nested clade phylogeographical analysis (NCPA) and analysis of molecular variance (amova), to examine 781 bp of mitochondrial DNA (nad4 and two tRNAs) from 82 individuals of P. mcallii. We tested whether populations of this species were recently connected, or if they were historically isolated prior to human modification of the region. Our NCPA results indicated significant population structure associated with the Colorado River, suggesting limited gene flow and potential isolation across this barrier. Populations west of the Colorado River, currently isolated from one another by human development, show less genetic differentiation. We also collected homologous sequence data from 34 individuals of P. platyrhinos and seven specimens morphologically intermediate between P. mcallii and P. platyrhinos, as a preliminary investigation of hybridization between these two species. From phylogenetic results of these data, we identified a species (Phrynosoma goodei) previously recognized as a subspecies of P. platyrhinos. Six of the morphologically intermediate specimens shared mtDNA haplotypes with P. goodei, while one was nested among P. mcallii haplotypes.