BACKGROUND: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides. RESULTS: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides. CONCLUSION: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides.