Emergence of new Puccinia striiformis f. sp. tritici races that overcome resistance of wheat cultivars is a challenging issue for wheat production. Although sexual reproduction of the fungus on barberry plants under field conditions in the spring in China has been reported, the diversity of the pathogen on barberry plants and the relationship to the population in wheat fields have not been determined. In the present study, two P. striiformis f. sp. tritici populations collected in western Shaanxi Province in May 2016, one from barberry plants (103 isolates) and the other from nearby wheat crops (107 isolates), were phenotyped for virulence and genotyped with simple sequence repeat (SSR) markers. The phenotypic and genotypic data of the two populations were compared to determine their relationships. A total of 120 races, including 29 previously known races (seven were shared by the two populations) and 91 new races (35 from barberry and 56 from wheat), were identified. Similarly, a total of 132 multilocus genotypes, including 51 only from barberry, 77 only from wheat, and four from both, were detected using the SSR markers. Analyses of molecular variance identified high (93%) genetic variance within populations and low but still significant variance (7%) between the populations. Nonparametric multivariate discriminant analysis of principal components and STRUCTURE analysis showed that the two populations had a close relationship with little genetic differentiation (F_ST = 0.038) and strong gene flow (Nm = 6.34, P = 0.001) between them. Although the analysis of linkage disequilibrium indicated clonal populations, the isolation of P. striiformis f. sp. tritici from barberry plants and the high genetic diversities in the barberry and wheat populations suggest that barberry plants provide aeciospores to infect wheat crops in the area. The information is useful for understanding stripe rust epidemiology and management of the disease.