The virulence analysis of Puccinia stiiformis f. sp. tritici (Pst), the cause of wheat stripe rust, is essential for predicting and managing the disease epidemic in Southwest China, where the wheat cultivation has significantly reduced in the past few decades due to the impact of this disease. From 2020 to 2021, 196 Pst isolates were collected from Guizhou, Yunnan, and Sichuan. The virulence and race assessments were conducted using Chinese differential genotypes. Additionally, the resistance expression of 102 wheat lines was evaluated in 2021 in two disease nurseries located in Ningnan and Jiangyou. All the 45 Pst isolates from Guizhou and Yunnan belonged to pathogroup Hybrid 46, with 36 identified as race CYR32. Among the 69 isolates from the Liangshan Prefecture, 67 belonged to the Hybrid 46 group, while the remaining two were identified as race CYR34 in the G-22 group. Furthermore, all 79 isolates from the western Sichuan Basin belonged to the G-22 group, with 54 identified as race CYR34. The diversity indices of the Pst populations from Guizhou, Sichuan, and Yunnan exhibited a sequential decline. Virulence variation among the Pst populations from Yunnan, Guizhou, and the Ganzi-Liangshan region was minimal; however, significant virulence differences were observed when these populations were compared to those from the western Sichuan Basin. Results from disease nurseries indicated that Pst virulence was notably stronger in Ningnan compared to that in Jiangyou. The Sichuan Basin exhibits a notable diversity in Pst virulence, coupled with a more frequent genetic exchange occurring between the Liangshan Prefecture and the Yunnan-Guizhou Plateau. This information is essential for developing effective management strategies to mitigate the impact of wheat stripe rust in this region.