Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important cereal fungal disease worldwide, including in the major wheat‐producing areas in China. In 2019, there was a widespread epidemic of the disease in the middle and lower reaches of the Yangtze River (MLRYR) area, including Zhejiang, Jiangsu, Anhui and Guangxi. However, in north‐western China, the postulated inoculum source of MLRYR, disease occurred mildly. Therefore, the origin of the Pst inoculum source for the MLRYR epidemic is still an open question. To address it, we collected 210 Pst uredinium samples from the Yangtze River basin and analysed their genetic structure with 13 pairs of simple‐sequence repeat (SSR) markers that we coupled with an upper airflow trajectory analysis. Our results suggest that the autumn inoculum sources of wheat stripe rust in MLRYR in 2019 probably came from south‐western over‐summering areas of Pst through long‐distance migration. We also found that Jingzhou and adjacent areas in Hubei Province function as an important bridge or stopover for inoculum sources.