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Molecular markers were used to study the genetic diversity, structure, and relationship of Juglans L. with nine populations (five from Juglans regia L. and four from Juglans sigillata Dode) in central and southwestern China. A moderate level of genetic diversity was observed at the population level with the number of effect alleles per locus (A E) ranging from 1.75 to 3.35 (average 2.39) and the proportion of polymorphic loci (P) equaling 100.0%. The expected heterozygosity (H E) within populations ranged from 0.389 to 0.687, and the average was 0.525. The proportion of genetic variation presented among populations accounted for 18.6% of the total genetic diversity. The overall gene flow (N m) among populations equaled 1.10. The unweighted pair-group method using arithmetic averages (UPGMA) clustering and the Mantel test showed that genetic distances among the nine populations are in a good agreement with their geographic distribution, supporting the viewpoint that J. regia and J. sigillata belong to one species. We suggest that the central area of the southwestern mountain regions of China could be considered as a priority for walnut genetic resource conservation.