Abstract: The Pallas’s cat (Otocolobus manul) is one of the most understudied taxa in the Felidae family. The species is currently assessed as being of “Least Concern” in the IUCN Red List, but this assessment is based on incomplete data. Additional ecological and genetic information is necessary for the long-term in situ and ex situ conservation of this species. We identified 29 microsatellite loci with sufficient diversity to enable studies into the individual identification, population structure, and phylogeography of Pallas’s cats. These microsatellites were genotyped on six wild Pallas’s cats from the Tibet Autonomous Region and Mongolia and ten cats from a United States zoo-managed population that originated in Russia and Mongolia. Additionally, we examined diversity in a 91 bp segment of the mitochondrial 12S ribosomal RNA (MT-RNR1) locus and a hypoxia-related gene, endothelial PAS domain protein 1 (EPAS1). Based on the microsatellite and MT-RNR1 loci, we established that the Pallas’s cat displays moderate genetic diversity. Intriguingly, we found that the Pallas’s cats had one unique nonsynonymous substitution in EPAS1 not present in snow leopards (Panthera uncia) or domestic cats (Felis catus). The analysis of the zoo-managed population indicated reduced genetic diversity compared to wild individuals. The genetic information from this study is a valuable resource for future research into and the conservation of the Pallas’s cat.