Overreliance on tribenuron has resulted in resistance evolution in waterstarwort. This study investigates the resistance mechanisms to tribenuron inwater starwort populations from China. The cytochrome P450 monooxygenase(P450) inhibitor malathion increased tribenuron sensitivity in allpopulations. The decrease in the amount of herbicide dose that causes 50%growth reduction (GR50) for the sensitive (S) population JS24 andthe resistant (R) populations JS16 and JS17 were 2.3-, 2.5-, and 4.1-fold,respectively. However, the GR50 values for the R populations werestill much higher than those of the S population. This observation indicatesthat P450-mediated enhanced metabolism is one mechanism for resistance inwater starwort. The glutathione-S-transferase (GST) activity could beinduced by tribenuron for all tested populations. In particular, the GSTactivity of JS16 is inherently greater and is more rapidly induced than thatof JS17 or JS24. Resistance attributed to mutant acetolactate synthase (ALS)alleles was identified by sequence analysis for each population. Pro197Sersubstitution was detected in JS16 and JS17. Molecular markers were alsodeveloped to rapidly identify resistance as well as individuals carrying thespecific Pro197Ser mutation in water starwort populations. The resistancepatterns experiment revealed that the R populations exhibited differentlevels of resistance to pyrithiobac sodium salt, florasulam, pyroxsulam, andflucarbazone-Na; however, R populations were sensitive to imazethapyr,fluroxypyr-meptyl, 2,4-D butylate, isoproturon, and diflufenican. This studyestablishes that either one or at least two resistance mechanisms areinvolved in herbicide resistance in water starwort. Moreover, thesemechanisms might contribute to the different levels of resistance totribenuron among water starwort populations.