AbstractHerbicide options for selective control of monocot weeds in rice (Oryza sativa L.) have historically been limited to a few modes of action such as inhibitors of acetolactate synthase (e.g., penoxsulam, imazamox), photosystem II (e.g., propanil), and acetyl-CoA carboxylase (e.g., cyhalofop). Florpyrauxifen-benzyl (Rinskor™) is a synthetic auxin molecule introduced to the U.S. rice herbicide market in 2018, providing broad-spectrum weed control (monocots and dicots), including hard-to-control species such as barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.], along with postemergence rice selectivity at very low use rates. Within the year of commercialization, field agronomists and academics identified E. crus-galli escapes in some areas where florpyrauxifen-benzyl had been sprayed. Further evaluation under controlled environments confirmed that those plants were able to survive florpyrauxifen-benzyl application at the label rate. Here, we identify the mechanism of resistance to florpyrauxifen-benzyl and penoxsulam in two E. crus-galli populations from Arkansas (AR-27) and Missouri (MO-18). Using high-resolution mass spectrometry, we compared the two resistant biotypes with known susceptible plants regarding their ability to metabolize florpyrauxifen-benzyl, florpyrauxifen-acid, and penoxsulam in planta. We discovered that the resistant plants share a common resistance mechanism to florpyrauxifen-benzyl and penoxsulam, involving hydrolysis of a methoxy group (likely mediated by a cytochrome P450 monooxygenase) followed by glucose conjugation. Given that penoxsulam has been widely used in rice fields for the past decade, these data suggest that some populations of E. crus-galli may have evolved resistance before the commercialization of florpyrauxifen-benzyl.