Understanding the origins and migration routes of phytopathogen inoculum is essential in predicting disease development and formulating control strategies. Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, is an airborne fungal pathogen threatening wheat production by long-distance migration. Due to large variation in geographic features, climatic conditions, and wheat production systems, inter-regional Pst dispersal routes in China remain largely unknown. In the present research, we sequenced 154 Pst isolates sampled from all the major wheat-growing regions in China to study the Pst population structure. Western Qinling Mountains, Himalayan region, and Guizhou Plateau were found to be centers of Pst origin in China. Combined with trajectory tracking and field disease surveys, long-distance Pst migration routes from individual origins were proposed. The present findings will improve current understanding of Pst origin and migration in China and emphasize the need for managing stripe rust at the national scale.