Ozone (O3) concentrations in periurban areas in East Asia are sufficiently high to decrease crop yield. However, little is known about the genotypic differences in O3 sensitivity in winter wheat in relation to year of cultivar release. This paper reports genotypic variations in O3 sensitivity in 20 winter wheat cultivars released over the past 60 years in China highlighting O3-induced mechanisms. Wheat plants were exposed to elevated O3 (82 ppb O3, 7 h day−1) or charcoal-filtered air (<5 ppb O3) for 21 days in open top chambers. Responses to O3 were assessed by the levels of antioxidative activities, protein alteration, membrane lipid peroxidation, gas exchange, leaf chlorophyll, dark respiration and growth. We found that O3 significantly reduced foliar ascorbate (−14%) and soluble protein (−22%), but increased peroxidase activity (+46%) and malondialdehyde (+38%). Elevated O3 depressed light saturated net photosynthetic rate (−24%), stomatal conductance (−8%) and total chlorophyll (−11%), while stimulated dark respiration (+28%) and intercellular CO2 concentration (+39%). O3 also reduced overall plant growth, but to a greater extent in root (−32%) than in shoot (−17%) biomass. There was significant genotypic variation in potential sensitivity to O3 that did not correlate to observed O3 tolerance. Sensitivity to O3 in cultivars of winter wheat progressed with year of release and correlated with stomatal conductance and dark respiration in O3-exposed plants. O3-induced loss in photosynthetic rate was attributed primarily to impaired activity of mesophyll cells and loss of integrity of cellular membrane as evidenced by increased intercellular CO2 concentration and lipid peroxidation. Our findings demonstrated that higher sensitivity to O3 in the more recently released cultivars was induced by higher stomatal conductance, larger reduction in antioxidative capacity and lower levels of dark respiration leading to higher oxidative damage to proteins and integrity of cellular membranes.