Tribenuron-methyl has been used widely for the last 15 yr to control white mustard in cereal crops from southern Spain. Since 2007, several cases of tribenuron-methyl resistance have been reported in wheat fields. Greenhouse and laboratory studies were conducted to characterize the mechanism of suspected tribenuron-methyl resistance in a white mustard biotype (hereafter AR16) from Malaga (southern Spain). Under greenhouse conditions, the dose (g ai ha−1) inhibiting fresh weight by 50% (ED50) was 5.20 and 0.57 for the AR16 and AR3 (known susceptible) biotypes, respectively. With the use of 14C-tribenuron-methyl, absorption and translocation from treated leaves were similar between biotypes. Thin-layer chromatography indicated foliar metabolism of tribenuron-methyl was low in both R and S biotypes. Assays on the binding affinity of tribenuron-methyl on acetolactate synthase (ALS) revealed enzyme activity was reduced by 50% (I50 value) at 638.7 and 0.23 nM for the AR16 and AR3 biotypes, respectively. This resulted in 2,777-fold greater resistance to tribenuron-methyl for the AR16 compared to AR3 biotype. Sequencing the gene encoding ALS, a proline/serine amino-acid substitution, was detected in position 197 of the A domain. Based on these results, it is concluded that tribenuron-methyl resistance in the AR16 biotype is due to a target-site mutation in the ALS enzyme, resulting in a lack of affinity to tribenuron-methyl.