Abstract: From all known Juglans regia MboI genomic BAC clones in NCBI database, 22 740 end sequences (BES) were downloaded and analyzed. A total of 4 732 SSRs (microsatellites) were found by using the MISA program. The frequency of SSRs was approximately 1/2.8 kb. Mono-to tri-nucleotide types accounted for 96. 86% of all the single SSRs in the BES. A/ T, AT/ AT and ATT/ AAT motifs were the repeat categories of most abundant mono-, di-and tri-nucleotide respectively. Fifty SSR primer pairs with different types and number of repeats were designed and synthesized, and 33 highly polymorphic SSR primer pairs were screened. Nineteen out of the 33 primer pairs had 1 or 2 alleles without nonspecific amplification. Those primer pairs were used to amplify SSR for 20 genotypes in the genus Juglans. The results showed that 2 - 9 polymorphic loci, 5.4 on average, were amplified per pair of primers. Polymorphism information content was greater than 0.5 in 17 SSRs loci with a mean value of 0.662 in all loci, which showed high polymorphism. The cluster analysis showed that the groups were clustered in accordance with species and origin. Our findings indicated that BES-SSRs identified in J. regia had high polymorphism and were new SSR primers for genetic analysis of Juglans.