Plants of 16 Italian cultivars of durum wheat at the tillering stage were exposed to a single pulse of ozone (O3, 200 ppb, 5 h) to reveal the mechanisms explaining their O3 tolerance/sensitivity by considering some leaf physiochemical traits [e.g., stomatal conductance, photosystem II (PSII) efficiency, antioxidant enzyme activity, and phytohormone synthesis]. At the end of the recovery period (48 h from the beginning of the exposure), all cultivars showed visible foliar injury in the form of widespread chlorosis which developed in bifacial ivory necrotic lesions scattered among the leaf veins of completely expanded leaves. Mongibello leaves showed not only the highest severity of visible injury (McKinney index = 62%), but also a reduction of PSII performance (Fv/Fm ratio: −22% compared with controls) together with an activation of the dissipation of the excess excitation energy as heat (qNP: +35%). The photosynthetic impairment observed throughout the whole experiment, was likely due to stomatal limitations (gs: −36% as average) and indicated that this cultivar was very sensitive to a pulse of O3. Similar results were obtained for Latino and Meridiano. Conversely, Iride, Orobel, and Portorico showed the lowest severity of visible injury (McKinney index around 13%) suggesting that they could be considered tolerant to a pulse of O3. These cultivars showed none or only transient changes due to O3 in terms of photosynthetic activity (e.g., A values), while the slight decrease of Fv/Fm ratio did not determine the photoinhibition of the PSII activity, since mechanisms for dissipating excess excitation energy were being activated. Ozone treatment induced a significant reduction of CO2 assimilation rate (−34%) in Orobel and this is not attributable to stomatal and non‐stomatal limitations. The remaining cultivars were considered moderately sensitive to O3, with McKinney index in the range of 20–43%. The measured biochemical traits are not decisive in determining the O3 sensitivity. In particular, the activity of phenylalanine ammonia lyase increased only in seven cultivars; among them, only in Latino, Vetrodur, and Simeto, this enhancement reflected in an accumulation of phenolic compounds suggesting potential higher protection against reactive oxygen species. This work highlights the relevance of identifying O3‐sensitive cultivars, in the optic to develop sustainable strategies to mitigate the impact of peak O3 episodes on wheat production.