Abstract: Bretschneidera sinensis Hemsl. is a model plant for research on angiosperm phylogeny, paleogeography, and paleoclimatology. To develop simple sequence repeat(SSR) markers for studies on population genetics of B. sinensis, candidate SSR loci were searched based on high-throughput sequencing and characterized using bioinformatics. Furthermore, SSR primer locus polymorphism in 12 genotypes without affinities was detected by simple sequence repeats with tailed primers. A total of 8772 SSR loci were obtained, which were distributed in 6732 unigenes. The appearance frequency and distribution density of SSR loci were 25.48% and 1/4.39 kb, respectively. In all SSR loci, single nucleotides, dinucleotides, and trinucleotides were the main repeat motif types, accounting for 53.72%, 29.42%, and 15.42% of total SSR loci, respectively. AG/CT was the most abundant dinucleotide repeat motif, accounting for 22.21% of total SSR loci. AAG/CTT was the most abundant trinucleotide repeat motif, followed by ACC/GGT and ATC/ATG. Of the 120 primers, the target fragment was detected in 68 pairs of primer products and 49 were successfully amplified in all tested genotypes and were verified as polymorphic, with the proportion of polymorphic loci being 72.06%. The number of alleles per locus ranged from 2 to 7 (average of 3.55). These results suggest that the SSR loci developed from on transcriptome sequence of B. sinensis were highly polymorphic. Analysis of the distribution characteristics of the high-throughput transcriptome sequence of B. sinensis could be helpful in gene mining, and these new polymorphic SSR markers should provide a valuable molecular tool for investigating the genetic diversity, population structure, phylogeographic history, and construction of core collection for ex situ conservation of this species.