In order to grasp the germplasm resources of walnut in Yunnan province, the genetic diversity and relationship of 21 walnuts varieties commonly planted in Yunnan province were analyzed using SSR fluorescent labeling technology. The results showed that the average number of allelic loci of 12 pairs of primers used in 21 walnut species samples was 7.916 7. The mean number of alleles, Shannon Wiener index (I) and polymorphic information content (PIC) per locus were 4.3516, 1.643 6 and 0.705 5. Twelve pairs of polymorphism information content (PIC) were higher than 0.25, indicating that the 12 pairs of primers had good polymorphism in the samples. In the phylogenetic analysis, the 21 walnut varieties were divided into 7 subgroups, indicating that the 21 walnut varieties had rich genetic diversity, extensive gene exchange and complex genetic background in the breeding process.