To exploit the walnut (Juglans regia) germplasm in Shennongjia Nature Reserve, the genetic diversity and genetic structure of walnut populations were studied based on 12 SSR markers. 108 alleles from 90 walnut trees of three walnut populations were amplified, and the proportion of polymorphism was 100%. The fact that level of total expectation heterozygosity (He) was 0.604, showed a moderate genetic diversity, and suggested a high probability of rare germplasm in the populations. Analysis from molecular variance revealed a significant genetic variation (Fst=0.082 2) among populations of walnut, which indicated a moderate differentiation of populations, this might cause by geographic isolation on the three populations, and some locus also suggested a heterozygous deficiency among all the samples. The cluster results showed that the genetic distances among the three populations were consistent with their geographic distance.