The developing seeds of Tung tree (Vernicia fordii) cultivar ‘Duiniantong’ were used to construct the cDNA library and a total of 3202 original EST sequences were generated. After removing redundant and poor quality sequences, 1047 non-redundant ESTs were detected to contain 212 SSRs by MISA. The dinucleotide and trinucleotide repeats were the most abundant SSRs, accounting for 68.39% and 25.94%, respectively. Furthermore, AG/CT was the most abundant repeat motif (43.87%), followed by AT/TA (19.34%) and AGC/GCT (5.66%). A total of 68 EST-SSR primer pairs corresponding to 68 ESTs were designed, of which 14 were identified as polymorphic SSR markers by PCR and data analyses. Application of the 14 SSRs in genetics anlysis of 169 V. fordii accessions revealed that a total of 41 putative alleles were generated with a mean of 2.93 alleles per locus. The expected heterozygosity (He) and the polymorphism information content (PIC) values ranged from 0.08 to 0.63 and 0.08 to 0.56, with a mean of 0.33 and 0.30, respectively. The 14 EST-SSR markers exhibited a moderate level of polymorphism in V. fordii accessions. The genetic similarity coefficients and the genetic distances varied from 0.9604 to 0.9986 and 0.0022 to 0.0404, respectively, indicating close genetic relationships among V. fordii populations. The genetic similarity-based dendrogram revealed that there was not a significant geographic pattern of genotypes across the collection areas of 169 V. fordii resouces.