This study documents the physiology and genetics of evolved atrazine resistance in a wild radish population from Western Australia. Plant response to atrazine treatment confirmed a high level of resistance in population WARR5. At 0.25 kg atrazine/ha, all plants from a susceptible population were killed, whereas resistant WARR5 was unaffected at the highest dose tested (4 kg atrazine/ha). Leaf photosynthesis in susceptible plants was inhibited after 1 kg atrazine/ha treatment, whereas leaf photosynthesis in WARR5 plants was unaffected. Furthermore, atrazine resistance was maternally inherited. Sequencing of a psbA gene fragment in resistant WARR5 and susceptible plants revealed a single point mutation resulting in a coding change from Ser264 to Gly of the D1 protein in resistant plants. We are confident that this mutation is the basis of resistance to the photosystem II inhibitors in this wild radish population.