We developed polymorphic microsatellite markers in Allium mongolicum Regel, a desert plant widely distributed in western China. To better conserve this species, we need to investigate its genetic diversity and population genetic structure, as well as its evolutionary history. Using the combined biotin capture technique, we isolated 13 novel polymorphic microsatellite loci and evaluated their characteristics using 60 individuals from two populations of A. mongolicum. Two to fourteen alleles per locus were identified for these microsatellites. The observed and expected heterozygosities ranged from 0.132 to 0.875 and 0.447 to 0.986, respectively. These microsatellite markers can be used to assess the genetic variation and genetic structure of A. mongolicum.