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Background: Resistance to 2,4‐Dichlorophenoxyacetic acid (2,4‐D) has been reported in several weed species since the 1950s; however, a biotype of Conyza sumatrensis showing a novel physiology of the rapid response minutes after herbicide application was reported in 2017. The objective of this research was to investigate the mechanisms of resistance and identify transcripts associated with the rapid physiological response of C. sumatrensis to 2,4‐D herbicide. Results: Differences were found in 2,4‐D absorption between the resistant and susceptible biotypes. Herbicide translocation was reduced in the resistant biotype compared to the susceptible. In resistant plants 98.8% of [14C] 2,4‐D was found in the treated leaf, whereas ≈13% translocated to other plant parts in the susceptible biotype at 96 h after treatment. Resistant plants did not metabolize [14C] 2,4‐D and had only intact [14C] 2,4‐D at 96 h after application, whereas susceptible plants metabolized [14C] 2,4‐D into four detected metabolites, consistent with reversible conjugation metabolites found in other 2,4‐D sensitive plant species. Pre‐treatment with the cytochrome P450 inhibitor malathion did not enhance 2,4‐D sensitivity in either biotype. Following treatment with 2,4‐D, resistant plants showed increased expression of transcripts within plant defense response and hypersensitivity pathways, whereas both sensitive and resistant plants showed increased expression of auxin‐response transcripts. Conclusion: Our results demonstrate that reduced 2,4‐D translocation contributes to resistance in the C. sumatrensis biotype. The reduction in 2,4‐D transport is likely to be a consequence of the rapid physiological response to 2,4‐D in resistant C. sumatrensis. Resistant plants had increased expression of auxin‐responsive transcripts, indicating that a target‐site mechanism is unlikely. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]