Abstract:A total of 48501 expressed sequence tags (ESTs) of Castanea mollissima were obtained from database of Fagaceae Genomics, of which 8479 ESTs contain 12116 simple sequence repeats (SSRs) with motifs longer than 10 bp, with the frequency of SSRs up to 24.98%. Dinucleotide and trinucleotide repeats are the main EST-SSR types in Chinese chestnut, accounting for 38.05% and 42.20% of all EST-SSRs, respectively. The Chinese chestnut EST-SSR markers showed 100% transferability across the two endemic species to China, C. henryi and C. seguinii. There were 106, 108 and 101 polymorphic loci from 115 loci in C. mollissima, C. seguinii and C. henryi, respectively. While 260 out of the 575 alleles (45.22%) were shared among the three species, unique alleles in each species were also detected. These informative EST-SSR markers can be used in population genetic studies and comparative mapping of Chinese chestnuts.