The objective of this study is to develop EST-SSR markers for further molecular evaluation of genetic resource for G. sinensis. Based on the transcriptome RNA-seq data from NCBI, a total of 41003 Unigenes were obtained by assembling the RNA-seq data of G. sinensis. The total length, average length and N50 of the Unigenes were 70.4 Mb, 1716 bp and 2533 bp, respectively. 8494 EST-SSR loci were detected in 7009 Unigenes, among which 1200 Unigenes contained two or more SSRs and 369 Unigenes contained compound SSRs. For all EST-SSR loci, 6494 primers were designed and 60 from them were randomly selected to validate their effectiveness. Consequently, 44 primer pairs can amplify target products, among which 17 primer pairs showed polymorphism and most polymorphism markers were identified to be located in UTR region. Furthermore, The PIC value ranged from 0.195 to 0.742, with an average of 0.501. Finally, we validated whether the primers could amplified target products in relatives of G. sinensis, among the 44 effective primers, 32, 31, 23, 24, 7, 40, 25, 18, and 18 primer pairs can amplified target products in G. tricanthos, G. japonica, G. japonica var. velutina, G. japonica var. delavayi, G. fera, G. australis, G. microphylla, Gymnocladus chinensis, Gymnocladus dioica, indicating the high transferability of our developed primers. The result demonstrated that mining EST-SSR loci using transcriptome data is one of the efficient ways for developing molecular markers in tree species, with the advantages of steady ampilification, high polymorphism and high transferability.