BACKGROUND: Sisymbrium orientale has evolved resistance to 2,4-D in Australia due to a 27 bp deletion in SoIAA2. However, one population of Sisymbrium orientale resistant to 2,4-D (R1) did not contain the SoIAA2 RESULTS: R1 is resistant to 2,4-D, 2-methyl-4-chlorophenoxyacetic acid (MCPA), and fluroxypyr with increased tolerance to dicamba, triclopyr and picloram. The 2,4-D resistance level for R1 is lower than resistant populations containing a 27 bp deletion in IAA2. 2,4-D resistance in R1 was due to a single dominant gene. Pretreatment of R1 with the P450 inhibitor malathion did not reduce resistance to 2,4-D. We identified a point mutation, Leu 175 Pro, in SoIAA34 in R1, but not in three susceptible (S) populations. This mutation was present (either homozygous or heterozygous) in all 2,4-D resistant plants in a segregating F2 population generated from a cross between S and R1 plants. Transgenic Arabidopsis plants expressing SoIAA34-R had significantly greater root length and dry mass than the wild-type (WT) or transgenic Arabidopsis plants carrying SoIAA34-S under 2,4-D treatment. CONCLUSION: We confirmed resistance to 2,4-D in the R1 population was controlled by a single dominant locus. Further, we verified that the Leu 175 Pro mutation in SoIAA34 is responsible for the 2,4-D resistance in R1. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.