Walnut (Juglans regia L.) is the most important species in the genus Juglans due to the high commercial value of its nuts and timber. Simple sequence repeats (SSRs) are considered as the markers of choice owing to their codominant nature. Since very few SSRs have been developed in walnut, this study aimed to develop numerous polymorphic SSRs from Class I repeats by using DNA sequences of the Chandler cultivar. In all, 800 SSRs were designed and tested in 8 Turkish, 3 French, and 5 US walnut cultivars, of which 88 (11%) did not produce bands, 161 (20.1%) were monomorphic, and 551 (68.9%) were polymorphic. In all, 2696 alleles were produced by the 551 polymorphic SSR loci in the 16 walnut cultivars, ranging from 2 to 14 alleles per locus with an average number of 4.9. Polymorphism information content ranged from 0.21 to 0.89 with an average of 0.62. Cluster analysis produced a very robust dendrogram. The walnut cultivars were separated into two main groups: all Turkish cultivars were included in one group, whereas the US and French cultivars were included in the other group. A set of 20 SSRs was selected for their high genetic diversity values to be used in further genetic studies in walnut. The novel SSR markers developed in this study could be used in future studies for constructing a genetic linkage map, analyzing population genetics, identifying parents, and conducting marker-assisted breeding, fingerprinting, and germplasm characterization in walnut.