Pine wilt disease is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, and is a chronic problem to pine forests (Pinus thunbergii and Pinus densiflora) in Japan. To promote more forward genetics for detecting quantitative resistance loci and to enhance resistance breeding programmes using marker assisted selection (MAS) or gene assisted selection (GAS), we have prepared genetic resources such as complementary DNA libraries, expressed sequences tags (ESTs), simple sequence repeat (SSR) markers, and a genetic linkage map. In this study, we report the development of EST-SSR markers from leaf and stem tissues cDNA libraries of P. thunbergii. A total RNA extracted from leaf and stem tissues of P. thunbergii was used for cDNA synthesis. ABI 3730xl-sequencing of cDNAs yielded raw EST sequences that were cleaned and assembled, and a total of 7,872 ESTs were obtained from the two cDNA samples. Among them, 756 non-redundant EST-SSR primer pairs were designed. As a first screening, PCR products from 8 P. thunbergii and 8 P. densiflora were screened for polymorphism. Results from first screening indicated that 25 % was not amplified, 43 % was monomorphic alleles, 28 % was polymorphic alleles, and 4 % was multi-locus alleles. Furthermore, polymorphic markers were further analyzed using 24 P. thunbergii and 24 P. densiflora to further investigate transferability and polymorphism of EST-SSR marker in P. thunbergii and P. densiflora.