One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm × J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profiles; the accessions with identical profiles appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identification, verification of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections.