The utility of intersimple sequence repeat (ISSR) markers for identification of English or Persian walnut (Juglans regia L.) cultivars was explored. Four cultivars were screened with 47 ISSR primers; eight of these primers, which generated reproducible and informative data, were selected for further study. Two individuals from each of 48 cultivars, including many currently important in the California walnut industry as well as accessions from Europe and Asia, were then examined with the eight ISSR primers. Polymerase chain reaction (PCR) products were separated on agarose gels and stained with ethidium bromide. Fifty-four bands were scored as present or absent in each cultivar; of these, 31 (57%) were polymorphic among the 48 cultivars. Combined data from the eight ISSR primers provided a unique fingerprint for each of the cultivars tested. Fifteen of the cultivars could be distinguished from all others with just one primer, 31 with a minimum of two primers, and two required three primers. Pairwise genetic distances between the cultivars were calculated and a dendrogram was generated using the neighbor-joining algorithm. Some of the groupings in the dendrogram corresponded to groups which, based on known pedigrees, are genealogically closely related. Others included accessions from diverse genetic and/or geographic origins. These results can be attributed to a combination of the limitations of the ISSR method for inferring genetic relationships, on the one hand, and the complex history of walnut cultivar development involving extensive exchange and breeding of germplasm from different geographic regions, on the other.