BACKGROUND: Diflufenican resistance has been reported in wild radish populations since 1998, but the resistance mechanisms have not been investigated. Recently, we identified a wild radish population (H2/10) from the Western Australian grain belt that is resistant (R) to the phytoene desaturase (PDS)-inhibiting herbicide diflufenican. RESULTS: Dose–response results showed this R population is 4.9-fold more resistant than the susceptible (S) population based on the LD50 R/S ratio. In addition, the R population also exhibits cross-resistance to the PDS-inhibiting herbicide fluridone. The cytochrome P450 inhibitor malathion reversed diflufenican resistance and partially reversed fluridone resistance in the R population. The full coding sequences of the PDS gene were cloned from the S and R plants and there are natural variations in the PDS gene transcripts/alleles with no correlation to resistance. In addition, the R plants had a level of PDS gene expression that is not significantly different from the S plants. CONCLUSION: These results demonstrated that diflufenican resistance in this R wild radish population is likely due to non-target-site based enhanced herbicide metabolism involving cytochrome P450s.