Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat in China. Due to the pandemic in 2017~2018, a total of 709 single-lesion samples, including 285 collected in 7 provinces in 2017 and 424 collected in 4 provinces in 2018, were isolated to explore the underlying causes. The population structure was analyzed by using 13 simple sequence repeat (SSR) makers. 306 multi-locus genotypes (MLGs) were detected and the genetic diversity was different among populations. Regardless of the year difference, 11 populations were clustered into two group: Gansu, Sichuan and Shaanxi belong to one group, while Yunnan, Guizhou, Hubei, and Anhui belong to another group. Genetic identity and shared genotypes along with the minimum spanning network (MSN) showed the Pst population from Sichuan play an intermediate role of genetic communication between Yunnan-Guizhou and Gansu. Since Berberis spp. has been identified as the alternate host of Pst, our results also provided a molecular evidence of potential sexual reproduction in Gansu population. In addition, the closer relationship between Yunnan-Guizhou and Hubei-Anhui was demonstrated. It was showed the role of Pst population in Yunnan-Guizhou might was underestimated in epidemiology. Previously, it was believed that the inoculum was limited locally. Our study provided strong evidence that the Pst inoculum source of Yunnan-Guizhou dispersed to Hubei areas by the upper airflow, and migrated to Anhui finally detriment Huang-huai regions.