[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 kb.Totally 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.
