BACKGROUND: Amaranthus tuberculatus (Moq.) Sauer (waterhemp) has emerged as one of several weed species that is resistant to synthetic auxin herbicides (SAHs). Among the mechanisms of resistance to SAHs, nontarget-site resistance (NTSR) has been of particular concern owing to its complexity. Here, we integrated linkage mapping with transcriptome analysis to explore NTSR mechanisms to two SAHs, dicamba and 2,4-D, in a multiple-herbicide-resistant A. tuberculatus population (CHR). RESULTS: Phenotypic evaluations of an F CONCLUSION: The results of this study demonstrated that resistance to dicamba and 2,4-D in the CHR population is under the control of genes at multiple loci. The weak phenotypic and genetic associations of resistance traits indicate that more than one NTSR mechanism confers resistance to dicamba and 2,4-D in this A. tuberculatus population. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.