Abstract Five thousand and twenty five ESTs of Juglans regia from the database of dbEST were screened using SSR-Hunter software, and 540 SSRs in 485 ESTs, accounting for 10.75% of ESTs were mined out. The SSR-ESTs occupied 9.7% of the total number of ESTs. Forty three primer pairs were designed for polymorphism analysis in 6 genotypes of J. regia, 1 genotype of J. nigra and 1 genotype of J. mandshurica. 40 primer pairs showed the amplification, accounting for 93% of designed primers, and 35 primer pairs showed polymorphisms, accounting for 87.5% of primers available. In the polymorphic 35 primer pairs, 27 primers showed polymorphisms in J. regia, the other 8 primers did not display polymorphism in J. regia, but showed polymorphisms among J. regia, J. nigra and J. mandshurica. Cluster analysis of the 8 genotypes was performed by UPGMA method, the result accords with the conventional taxonomy.
