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Flixweed (Descurainia sophia L.) is a problematic and widespread weed in winter wheat fields and has been controlled by tribenuron-methyl for more than twenty years in China. In this study, a flixweed accession (HB25) with an Asp-376-Glu mutation in acetohydroxy acid synthase (AHAS) was identified and purified. The purified HB25 accession (pHB25) developed 758.1-fold resistance to tribenuron-methyl and exhibited obvious cross-resistance to four AHAS-inhibiting herbicides. The R/S ratios of GR 50 to herbicides of halosulfuron-methyl (SU), flumetsulam (TP), imazethapyr (IMI) and pyribenzoxim (PTB) were 346.1 15.7, 8.1 and 7.1, respectively. The reduced AHAS sensitivities to four different AHAS-inhibiting herbicides, which were caused by the Asp-376-Glu mutation, were responsible for the resistance and cross-resistance to AHAS-inhibiting herbicides. The R/S ratios of I 50 to tribenuron-methyl, halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 844.5, 532.9, 74.5, 13.3 and 5.5 respectively. The results of AHAS activity in vitro were highly correlated with that of whole-plant response experiments.