Previous studies on the relationship of barberry (Berberis spp.) and wheat stem rust suggested that, although some barberry species can serve as alternate hosts for the stem rust fungus Puccinia graminis f. sp. tritici, barberry plants play no role in wheat stem rust development and virulence variation of P. graminis f. sp. tritici in China. In the present study, severe rust infections on Berberis shensiana, B. brachypoda, B. potaninii, B. soulieana, and B. aggregata were observed during field surveys in 2011 and 2012. Through artificial inoculation of wheat seedlings (‘Mingxian 169’) under greenhouse conditions with aeciospores from naturally infected barberry plants, 185 isolates of P. graminis f. sp. tritici were obtained. From the 27 selected isolates that were tested on a set of wheat genotypes used to differentiate P. graminis f. sp. tritici races in China, 18 races were identified, of which 8 races were new and others were of Chinese 21 and 34 race groups. In addition to the information of virulence or avirulence patterns on the Chinese differentials, none of the races were virulent to resistance gene Sr31. The virulence frequencies based on individual Sr genes or differentials ranged from 0 to 96%. Using molecular markers for P. graminis f. sp. tritici, 75 of 4,036 samples of single aecia were identified as P. graminis f. sp. tritici. The low detectable rate of P. graminis f. sp. tritici was verified based on both infection and molecular marker tests. This observation may be correlated with the low levels of wheat stem rust in the surveyed regions. This study shows that P. graminis f. sp. tritici can produce new races through sexual reproduction on Berberis spp. in China.