Iron walnut, Juglans sigillata Dode, restricted to southwestern China, has its centre of distribution in Yunnan Province which has a varied climate, geography and rich plant diversity. Yunnan contains abundant J. sigillata germplasm. In this study, a provincial-scale set of walnut germplasm resources (14 populations comprising 1122 individuals) was evaluated for genetic diversity based on 20 simple sequence repeat (SSR) loci. The number of SSR alleles per locus ranged from 7 to 27, with an average of 17.55. Mean allelic richness and mean private allelic richness ranged from 3.40 to 4.62 and 0.11 to 0.36, with average of 3.93 and 0.30, respectively. Expected heterozygosity (He) varied from 0.26 to 0.78, with an average of 0.57. Polymorphism information content ranged from 0.22 to 0.79, with an average of 0.57. Genetic differentiation (FST) was 0.05, indicating that only 5% of total genetic variability was inter-populational, a finding supported by an analysis of molecular variance and STRUCTURE analysis. Relatively high gene flow (Nm = 6.70) was observed between populations. A unweighted pair-group method with arithmetic analysis classified the 14 populations into two major groups. Mantel testing uncovered a significant correlation between geographic distance and genetic distance (r = 0.33, P = 0.04). Overall, the research revealed a moderately high level of genetic diversity in the germplasm and low genetic differentiation among populations, which showed great potential for further development and exploitation of this resource.