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Pedicularis rex C. B. Clarke ex Maxim., an endemic species with potential horticultural traits from Himalaya, has a unique cup-like petiole structure and highly infraspecific floral variation among members of the lousewort genus (Orobanchaceae). We developed 13 microsatellite markers from three microsatellite-enriched libraries (AG, AC, and AAG) of P. rex with a modified biotin–streptavidin capture technique. Polymorphism of each locus was assessed in 22 individuals with representation of five populations of P. rex. Number of alleles per locus (A) ranged from two to seven with an average of 4.38. The observed and expected heterozygosities varied from 0.03 to 0.86 and 0.45 to 0.84, respectively. Additionally, among the 13 identified microsatellite markers, 11 of them were successfully amplified in species P. thamnophila, and five of them showed polymorphisms. This study may provide important information for further investigation on the population genetics, introduction, and acclimatization of P. rex and its congeners.