© 2016 Society of Chemical IndustryBACKGROUND: When used at effective doses, weed resistance to auxinic herbicides has been slow to evolve when compared with other modes of action. Here we report the evolutionary response of a herbicide-susceptible population of wild radish (Raphanus raphanistrum L.) and confirm that sublethal doses of 2,4-dichlorophenoxyacetic acid (2,4-D) amine can lead to the rapid evolution of 2,4-D resistance and cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides. RESULTS: Following four generations of 2,4-D selection, the progeny of a herbicide-susceptible wild radish population evolved 2,4-D resistance, increasing the LD50 from 16 to 138 g ha-1. Along with 2,4-D resistance, cross-resistance to the ALS-inhibiting herbicides metosulam (4.0-fold) and chlorsulfuron (4.5-fold) was evident. Pretreatment of the 2,4-D-selected population with the cytochrome P450 inhibitor malathion restored chlorsulfuron to full efficacy, indicating that cross-resistance to chlorsulfuron was likely due to P450-catalysed enhanced rates of herbicide metabolism. CONCLUSION: This study is the first to confirm the rapid evolution of auxinic herbicide resistance through the use of low doses of 2,4-D and serves as a reminder that 2,4-D must always be used at highly effective doses. With the introduction of transgenic auxinic-herbicide-resistant crops in the Americas, there will be a marked increase in auxinic herbicide use and therefore the risk of resistance evolution. Auxinic herbicides should be used only at effective doses and with diversity if resistance is to remain a minimal issue. © 2016 Society of Chemical Industry.