Genetic diversity of 1061 walnut germplasm from 14 prefectures and cities of Yunnan Province was analyzed by SSR molecular marker technique. On the basis of this, a core collection was constructed through least distance stepwise sampling (LDSS) and tested for its diversity. The results showed that 322 alleles were detected by 19 pairs of SSR primers (on average 16.95 per pair of SSR primer). The means of Shannon’s information index (I), expected heterozygosity (He) and Nei’s gene diversity (Nei) were respectively 1.3375, 0.6040 and 0.6043, which indicated a relatively high genetic diversity of the germplasm. The genetic diversity distributed differently among the 14 prefectures and cities. Diqing Prefecture had the highest genetic diversity while Lincang had the lowest. Sampling rates of 30%-5% were taken for construction of the primary core collection, secondary core collection, core collection-1 and core collection-2, on the basis of UPGMA clustering results. With the genetic diversity analysis and t-test, the core collection-1 with 10.84% sampling rate and 115 samples was determined to be the core collection of walnut germplasm in Yunnan.