The valley of Cuatro Ciénegas, an aquatic oasis located in the Mexican Chihuahuan Desert, exhibits the highest level of endemism in North America and is a Mexican National Protected Area. However, little is known about the evolutionary distinctiveness of several vertebrate species present in the Cuatro Ciénegas valley. We conducted a phylogeographic study using mitochondrial haplotypes from the centrarchid fish Lepomis megalotis to determine if the populations found within the valley were evolutionarily distinct from populations outside the valley. We also examined if there was evidence of unique haplotypes of this sunfish within the valley. Genetic divergence of L. megalotis suggests populations within the valley are evolutionarily unique when compared to L. megalotis outside the valley. Significant mitochondrial sequence divergence was also discovered between L. megalotis populations on either side of the Sierra de San Marcos that bisects the valley. Our results reinforce previous studies that suggest the organisms occupying aquatic habitats not only within Cuatro Ciénegas but also in each of the two lobes of the valley generally deserve independent consideration during management decisions.