Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important disease on wheat, seriously threatening wheat production worldwide. China is one of the largest stripe rust epidemic regions in the world. The pathogen sexual reproduction and migration routes between Tibet and the other regions in China are still unknown. In this study, we obtained 961 Pst isolates from 1,391 wheat leaf samples from Gansu (277), Shaanxi (253), Sichuan (172), and Tibet (259), comprising 13 natural populations, and genotyped them with simple sequence repeat (SSR) markers. The isolates can be divided into two distinct clusters based on DAPC and STRUCTURE analyses. The genetic diversity of Longnan (in Gansu) and Yibin (in Sichuan) populations was the highest and lowest among the 13 populations, respectively. The hypothesis of multilocus linkage disequilibrium was rejected for the populations from Linzhi in the Himalayan, Longnan, Hanzhong, Guangyuan, Mianyang, Liangshan, and Chendu in the south Qinling Mountains at the level of P = 0.01, which indicated significant linkage among markers in these populations. Populations in the other regions had extensive gene exchange (Nm > 4); little gene exchange was found between Tibet and the other regions (Nm < 1). The results suggest that the Tibet epidemic region of Pst is highly differentiated from the other epidemic regions in China.