Flixweed [Descurainia sophia (L.) Webb ex Prantl] is a notorious broadleaf weed that is widely distributed in winter wheat–growing areas of China and has evolved resistance to tribenuron-methyl mainly due to target-site resistance (TSR) mutations in acetolactate synthase (ALS). In the current research, two ALS genes were identified in tribenuron-methyl–susceptible (TS) or tribenuron-methyl–resistant (TR) D. sophia. Resistance mutations of Asp-376-Glu and Pro-197-Ala were identified on ALS1 and ALS2 isozymes in TR D. sophia, respectively. The TR D. sophia evolved 10,836.3-fold resistance to tribenuron-methyl and displayed cross-resistance to multiple ALS-inhibiting herbicides with different chemical structures. Dose response experiments and ALS activity assay indicated that two mutated ALS isozymes contributed differentially in resistance to tribenuron-methyl, flucetosulfuron, and pyribenzoxim. In addition, the relative expression level of the ALS1 gene was 2.2- and 1.6-fold higher than ALS2 genes in TR D. sophia at 1 and 7 d after tribenuron-methyl treatment, respectively. In contrast, the relative expression level of ALS1 and ALS2 in TS D. sophia is similar. This is the first research that explored different roles of ALS isozymes in resistance to ALS-inhibiting herbicides, which might provide a new perspective for the weed resistance management.