Research was conducted to establish a method to investigate the resistance level of flixweed to tribenuron-methyl and the evolved biochemical resistance mechanism. Four resistant biotypes were collected from wheat fields in Mazhuangcun, Jiacun, Dishangcun, and Bafangcun in the Hebei province of China where tribenuron-methyl had been continuously used for more than 10 yr. Two susceptible biotypes were collected from wheat fields where tribenuron-methyl was never applied. Different biotypes were assessed by petri-dish bioassay, whole-plant bioassay, and acetolactate synthase (ALS) assay. Comparisons of data indicated a similarity between methods and that experiments demonstrated that petri-dish bioassay was a feasible method to identify flixweed resistant to tribenuron-methyl. Data indicated differences among the flixweed biotypes when assessed by the petri-dish bioassay, whole-plant bioassay, or ALS enzyme assay, and a close association was obtained for the three bioassay methods. ALS resistance varied by biotypes with Mazhuangcun > Jiacun > Dishangcun > Bafangcun. Target-site enzyme assay data indicated that the resistant biotype's enhanced ALS activity was the biochemical mechanism that induced flixweed's evolved resistance to tribenuron-methyl. The concentrations of tribenuron-methyl causing 50% inhibition of ALS activity of the four resistant biotypes were 1,359, 513, 184, and 164 nM; in the susceptible biotypes these concentrations were 64 and 65 nM. Resistance indexes were 21, 8, 3, and 3 for Mazhuangcun, Jiacun, Dishangcun, and Bafangcun biotypes, respectively.