The stripe rust pathogen Puccinia striiformis f. sp. tritici frequently causes significant yield losses in China, due to rapid development of new races that overcome resistance in wheat cultivars. Indirect evidence suggests that sexual reproduction occurs in the P. striiformis f. sp. tritici population in China but direct evidence was still lacking. In this study, a large-scale survey of barberry (Berberis spp.) was conducted in Gansu, Shaanxi, Tibet, and Xinjiang provinces in western China. In total, 9,297 single-aecial (SA) samples were used to inoculate a susceptible wheat cultivar to identify samples of P. striiformis f. sp. tritici. Sixteen of the SA samples were identified as P. striiformis f. sp. tritici. When tested on the wheat differentials for identifying P. striiformis f. sp. tritici races, 15 of the 16 SA samples had different virulence patterns, indicating that they were sexually produced through barberry. From the 16 SA samples, 118 single-uredinium (SU) isolates were obtained, from which 88 virulence patterns were identified when tested on 17 Yr single-gene lines. The virulence patterns had relatively narrow virulence spectra, ranging from 0 to 9, with a mean of four virulences per SU isolates. Of the 17 Yr genes, no virulences were detected for Yr5, Yr10, and Yr15; virulences to YrTr1, Yr24, and Yr27 were extremely low (<3%); those to YrSP, Yr9, Yr28, and Yr2 were low (13.6 to 28.0%); those to Yr7, Yr17, Yr8, and YrExp2 were moderate (33.1 to 48.3%); and those to Yr6, Yr44, and Yr25 were high (52.5 to 72.9%). This study provides direct evidence that natural sexual reproduction occurs in the P. striiformis f. sp. tritici population in China, but the frequency appears to be very low. The sexual reproduction on alternate host plants can generate a great virulence diversity, which may have contributed to the high variation in the P. striiformis f. sp. tritici population in China.