Walnut,Juglans regia L., has been cultivated for its edible nut and timber since ancient times. It is one of the most valuable and widely cultivated horticultural commodities in the world. Simple sequence repeats (SSRs) have a wide range of applications in plants, and there is still a necessity to develop novel SSR markers for each plant species. Bacterial artificial chromosome-end sequences (BES) are good sources to generate novel SSR markers. In pursuit of this objective, a total of 558 BES-SSR primer pairs were designed for J. regia. Of them, 507 (91%) had amplifications, and 307 were polymorphic when tested in eight walnut cultivars. A total of 1097 alleles were generated from 307 polymorphic SSR loci, ranging from two to eleven, with an average of 3.6 per locus. Polymorphism information contents (PIC) varied from 0.11 to 0.88 with an average of 0.46. Di-nucleotide motifs had higher polymorphism and genetic diversity values than the other motif types. The rate of polymorphism increased with the repeat length, therefore, class I SSRs are useful source of polymorphic DNA markers in walnut. Novel SSR markers developed in this study may possess potential applications such as fingerprinting, marker-assisted breeding, germplasm characterization and genetic linkage mapping in walnut and other species of Juglans. (C) 2016 Published by Elsevier B.V.