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Pupfishes (genus Cyprinodon) are iconic of biodiversity and endemism in the desert southwest of North America. Most of these species are imperiled, primarily because of excessive exploitation of water resources in this arid region. The critically endangered Julimes pupfish, Cyprinodon julimes, is restricted to a small, isolated, and highly modified desert spring in Chihuahua, Mexico. We evaluated effective population size (microsatellites) and genetic variation (microsatellites and mitochondrial DNA) to determine the conservation genetic status of this species. The effective population size was critically low and indicated that this pupfish is at genetic risk of extinction through loss of adaptive variance and, potentially, from inbreeding depression. Mitochondrial variation was also extremely low, and haplotype frequency was biased heavily in favor of one of two variants. The uncommon haplotype was derived from a past hybridization event with the closely related C. eximius; whether cessation of introgressive hybridization is relevant to conservation management of Julimes pupfish is unknown but may be important to consider. Minimally, C. julimes is compromised genetically. Baseline population genetic information provided by this study will be vital to long-term monitoring of this highly imperiled species.