Elm breeding programs worldwide have relied heavily on Asian elm germplasm, particularly Ulmus pumila , for the breeding of Dutch elm disease tolerant cultivars. However, the extent and patterning of genetic variation in Asian elm species is unknown. Therefore, the objective of this research was to determine the extent of genetic diversity among 53 U. pumila accessions collected throughout the People’s Republic of China. Using 23 microsatellite loci recently developed in the genus Ulmus , a total of 94 alleles were identified in 15 polymorphic and 4 monomorphic loci. The average number of alleles per locus was 4.9, with a range of 1–11 alleles. Gene diversity estimates per locus ranged from 0.08 to 0.87, and the non-exclusion probability for the 15 polymorphic loci combined was 0.7 × 10−9. Nineteen region-specific alleles were identified, and regional gene diversity estimates were moderately high (0.48–0.57). The genetic relationships among accessions and regions were estimated by UPGMA and principal coordinate analysis. Both techniques discriminated all accessions and regions. Two microsatellite markers (UR175 + UR123 or Ulm-3) were sufficient to discriminate up to 99.7% of the accessions studied. This research provides useful information for DNA-based fingerprinting, breeding, ecological studies, and diversity assessment of elm germplasm.