AbstractWheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), significantly impacts global wheat production, including China. Monitoring Pst virulence is crucial for disease management. This study used 18 Yr single‐gene isogenic lines of wheat to characterize the virulence of 2023 Pst collections in China. A total of 103 isolates were collected from 11 provinces, and 61 races were identified. Frequencies of virulence to the 18 Yr genes varied from 0% to 95.1%, with low frequencies (defined as less than 10%) to Yr85 (7.8%) and Yr10 (8.7%); moderate frequencies (10%–40%) to Yr24 (15.5%), Yr32 (16.5%), YrSP (37.9%) and Yr76 (37.9%); and high frequencies (>40%) to Yr7 (47.6%), Yr1 (68.0%), Yr9 (69.9%), Yr27 (69.9%), Yr17 (79.6%), Yr6 (89.3%), YrExp2 (92.2%), Yr43 (93.2%), Yr8 (95.1%) and Yr44 (95.1%). Yr6, Yr8, Yr17, Yr27, Yr43, Yr44 and YrExp2 have almost lost their effectiveness in all provinces, while Yr10, Yr24, Yr32 and Yr85 are effective in Hebei, Shandong, Shaanxi, Gansu and Anhui. No isolate was virulent to Yr5 or Yr15, indicating their effectiveness against Pst populations in China. The Pst isolates had a wide virulence spectrum ranging from 3 to 16 virulence factors, with an average of 9.5. Comparing 2023 to 2021, the PstCN‐040 and races sharing virulence to Yr1, Yr6, Yr7, Yr8, Yr9, Yr17, Yr43, Yr44 and YrExp2 predominated, with frequencies 16.4% and 25.2%. This study provides valuable information on the virulence composition of Pst populations in China, helping to guide the development of genetic resistance for wheat in China.