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Green foxtail [ Setaria viridis (L.) P.Beauv.] is a troublesome grass weed that is widely distributed in maize (Zea mays L.) fields across China. Many populations of S. viridis have evolved resistance to the acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron. The objectives of this research were to confirm nicosulfuron resistance in these populations and to investigate the basis of nicosulfuron resistance. Whole-plant dose-response experiments showed 6 out of 13 S. viridis populations were highly resistance (20–30 times) to nicosulfuron. Sequencing of the ALS gene revealed two amino acid mutations, Asp-376-Glu and Pro-197-Ala, in the nicosulfuron-resistant populations. A malathion pretreatment study revealed that the R376 and R197 subpopulations might have cytochrome P450s–mediated herbicide metabolic resistance. The resistant populations were cross-resistant to imazethapyr but sensitive to topramezone and quizalofop-p-ethyl. This is the first report of resistance to ALS inhibitors conferred by target site mutations (Asp-376-Glu or Pro-197-Ser) and possible cytochrome P450s-involved metabolism in S. viridis. [Display omitted] • Six Setaria viridis populations were highly resistant to nicosulfuron. • ALS sequencing revealed Asp-376-Glu and Pro-197-Ala mutations. • P450s inhibitor malathion could reduce nicosulfuron resistance in Setaria viridis. [ABSTRACT FROM AUTHOR]