The genetic structure and diversity of natural populations of Juglans regia L. in Iran were characterized using 11 microsatellite loci. A total of 105 individuals from seven populations were sampled. A high level of genetic diversity was observed within populations with the number of alleles per locus (A) ranging from three to 11 (average = 5.73), the proportion of polymorphic loci was 100%, and the expected heterozygosity ranged from 0.598 to 0.848 (average = 0.707). The proportion of genetic differentiation present among populations accounted for 12% of the total variation. Such considerable interpopulation differentiation detected in J. regia L. could have resulted from several factors, including restricted gene flow between populations. Significant departures from Hardy-Weinberg equilibrium were observed for WGA276, WGA32, and WGA321 loci. The deviations were primarily the result of the surplus of heterozygotes. The unweighted pair group method with arithmetic mean cluster analyses based on Nei's unbiased genetic distances separated the seven populations into two main groups.