Narrow-leafed lupin (Lupinus angustifolius L.) is an important grain legume crop in Australia. Metribuzin is an important herbicide used to control weeds in lupin crops. This study investigated metribuzin tolerance mechanism in narrow-leafed lupin by comparing two induced mutants (Tanjil-AZ-33 and Tanjil-AZ-55) of higher metribuzin tolerance with the susceptible wild type. Sequencing of the highly conserved region of the chloroplast psbA gene (target site) revealed that the sequences of the wild type and the mutants were identical and therefore metribuzin tolerance is not target site based. Photosynthetic activity was measured and the leaf photosynthesis of the two tolerant mutants was initially inhibited after metribuzin treatment, but recovered within 2.5 days whereas that of the susceptible plants remained inhibited. The photosynthetic measurements confirmed the target site chloroplast was susceptible and the tolerance mechanism is non-target site based. Investigation with known cytochrome P450 monooxygenase inhibitors (omethoate, malathion and phorate) showed that tolerance could be reversed in both mutants, indicating the tolerance mechanism in two tolerant mutants may involve cytochrome P450 enzymes. Interestingly, the inhibitor tridiphane reversed metribuzin tolerance of only one of the two tolerant mutants, indicating diversity in metribuzin tolerance mechanisms in narrow-leafed lupin. These results signify that further investigation of metribuzin metabolism in these plants is warranted. In conclusion, metribuzin tolerance mechanism in lupin mutants is non-target site based, likely involving P450-mediated metribuzin metabolism.