Various abiotic and biotic stressors affect crop and weed plant performance in agroecosystems. Ozone (O3) tolerance in plants is partly regulated by the genotype and phenotypical properties, and it varies greatly in related species of wild and crop backgrounds. Thus, a continuous increase in atmospheric O3 concentration could change population dynamics of sexually compatible crop and weed species, and consequently affect crop-to-wild gene flow in the future. One way to build resistance against a biotic stressor, in this case insect-mediated herbivory, in crop plants is transgene-mediated insecticidal toxin production. In this study we aimed to describe how the physiological and phenological responses in a crop Brassica and its weedy relatives functioned to affect their comparative O3 tolerance. Furthermore, we studied how harbouring a transgene affects these responses in B. napus and B. rapa × transgenic B. napus BC2F2 backcross hybrid plants to reveal any within-plant trade-offs among toxin production, growth and O3 tolerance. We found a higher number of O3 symptoms but more effective compensatory assimilate allocation directed to reproduction for wild B. rapa compared to crop B. napus under elevated O3. This result suggested that the invasion-orientated strategy of producing a high number of seeds when vegetative growth is limited might improve the performance of weedy species under elevated O3. The probabilities for crop-to-wild transgene flow could be increased through higher seed production in hybrids under elevated O3, but the germination of hybrid seeds in particular was hampered by O3. The presence of transgenes did not perturb fecundity, within-plant biomass allocation or O3 tolerance of B. napus.