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