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Amaranthus retroflexus L., a troublesome annual dicotyledonous weed species, is highly competitive with soybean (Glycine max L.). A single-dose herbicide-resistance screening assay identified an A. retroflexus population with suspected resistance to fomesafen. Whole-plant dose–response assays demonstrated that the resistant population (2492) was resistant to protoporphyrinogen oxidase (PPO)-inhibiting herbicides (50.6-fold fomesafen resistance and > 8.1-fold lactofen resistance) compared to a susceptible (S) population. PPX2 gene sequence analysis showed an Arg 128 Gly amino acid substitution in the 2492 population. Moreover, pretreatment of malathion and the fomesafen metabolic assays through HPLC–MS demonstrated enhanced fomesafen metabolism in the 2492 population. Additionally, the 2492 population was 10.4-fold more resistant to the ALS-inhibiting herbicide imazethapyr and 16.8-fold more resistant to thifensulfuron-methyl than the S population. ALS gene sequence analysis showed an Ala 205 Val amino acid substitution in the 2492 population. This population of A. retroflexus has coexisting target-site resistance and non-target-site mechanisms for resistance to fomesafen. Multiple herbicide resistance may mean it is necessary to adjust weed management strategies to better control the resistant population. [Display omitted] • A Amaranthus retroflexus L. population showed multiple resistance to ALS- and PPO-inhibiting herbicides. • Arg 128 Gly mutation in PPX2 and Ala 205 Val mutation in ALS were detected in this population. • Malathion pretreatment and HPLC-MS experiments indicated enhanced metabolism likely play a role. [ABSTRACT FROM AUTHOR]