Zelkova schneideriana Hand.-Mazz. of the Ulmaceae family is a Tertiary relict and economically deciduous tree species endemic to Central and Southern China. In this study, we performed a transcriptome sequencing of Z. schneideriana using high-throughput sequencing approach to detect polymorphic expressed sequence tag-simple sequence repeats (EST-SSR) markers. A total of 3,235 microsatellite loci were detected from 53,517 unigenes. A set of 30 microsatellite markers were randomly selected to validate in 41 individuals from three populations, of which 10 were polymorphic. The number of alleles per locus ranged from 3 to 11. The observed heterozygosity and expected heterozygosity ranged from 0.366 to 0.829 and 0.439 to 0.848, respectively. These polymorphic SSR primers showed good transferability across different Zelkova species, and are valuable for future studies on genetic diversity, conservation, phylogeography, and species delimitation in Z. schneideriana, as well as other Zelkova species.