Echinochloa phyllopogon (late watergrass) is a major weed of California rice (Oryza sativa) that has evolved cytochrome P450-mediated metabolic resistance to different herbicides with multiple modes of action. E. phyllopogon populations from Sacramento Valley rice fields have also recently shown resistance to the herbicide clomazone. Clomazone is a proherbicide that must be metabolized to 5-ketoclomazone, which is the active compound that inhibits deoxyxylulose 5-phosphate synthase, a key enzyme of the nonmevalonate isoprenoid pathway. This study evaluated the differential clomazone metabolism within strains of the same species to investigate whether enhanced oxidative metabolism also confers clomazone resistance in E. phyllopogon. Using reverse-phase liquid chromatography-tandem mass spectrometry techniques in the multireaction monitoring mode, we elucidated that oxidative biotransformations are involved as a mechanism of clomazone resistance in this species. E. phyllopogon plants hydroxylated mostly the isoxazolidinone ring of clomazone, and clomazone hydroxylation activity was greater in resistant than in susceptible plants. The major clomazone metabolites resulted from monohydroxylation and dihydroxylation of the isoxazolidinone ring. Resistant plants accumulated 6- to 12-fold more of the monohydroxylated metabolite than susceptible plants, while susceptible plants accumulated 2.5-fold more of the phytotoxic metabolite of clomazone, 5-ketoclomazone. Our results demonstrate that oxidative metabolism endows multiple-herbicide-resistant E. phyllopogon with cross-resistance to clomazone through enhanced herbicide degradation and lower accumulation of the toxic metabolite in resistant versus susceptible plants.