Abstract: A cDNA library of stem and leaf from Betula luminifera was constructed. The primary titer of cDNA library was about 1.5×106 pfu/mL, its recombinant efficiency reached 97.3%, and the size of insert DNA fragments ranged from 0.5 to 3.0 kb, with an average of 1.3 kb. The results indicated that it was a higherquality cDNA library, and could be used in gene cloning and gene expression profile analysis. Distribution and frequency of SSRs were analyzed in 224 nonredundant ESTs from B. luminifera cDNA library, using online searching software. The results showed that 60 SSRs distributed in 47 EST sequences, accounting for 26.80% of all ESTs. Dinucleotide would be the major repeat types, accounting for 70.00% of the total number of acquired SSRs. The trinucleotide and tetranucleotide repeats accounted for 28.30% and 1.70% respectively. This research might lay the foundation for designing the targeted ESTSSR primers and genetic diversity analysis by mining the information of EST-SSR loci in B. luminifera EST sequence data.