We used DNA sequences from several mitochondrial regions (ND2, ND4, and adjacent tRNAs) to examine genetic structure and divergence among populations of the Striped Plateau Lizard, Sceloporus virgatus. In the northern portion of this species' range, populations occupy mesic habitats at higher elevations in the mountain ranges of the Madrean Archipelago, and are thus isolated by dry, inhospitable valleys. High genetic differentiation among four of these ranges, and low genetic variation within ranges, imply that gene flow among them has been limited and isolation is ancient, having occurred hundreds of thousands or even millions of years ago. These results conflict with expectations derived from geographical and paleontological data that indicate ecological conditions favoring gene flow occurred as recently as 11,000 years ago.