In order to study the responses of winter wheat cultivars released in different years to short-term high O3 exposure, an old cultivar (‘Nongda 311’, released in 1960s) and a modern one (‘Yannong 19’, released in 1990s) were treated with an O3 exposure (145 ± 12 mm3 m−3, 4 h d−1 for 3 d) shortly after anthesis stage (> 50 % main stems blossomed). During the O3 exposure, light-saturated photosynthetic rate (PN) and stomatal conductance (gs) of both cultivars decreased considerably. Elevated O3 did not decrease dark-adapted maximum photochemical efficiency, but induced significant reduction in actual photochemical efficiency and thereby considerably increase in non-photochemical quenching. PN, gs of the modern cultivar ‘Yannong 19’ decreased more than the older one ‘Nongda 311’, indicating the former exhibited higher sensitivity to O3 than the latter. After O3 exposure, PN, gs and chlorophyll (Chl) content in flag leaf decreased more quickly than control, indicating induction of faster premature leaf senescence. As a result, the short-term O3 exposure caused substantial yield loss, with larger reduction in ‘Yannong 19’ (−19.2 %) than in ‘Nongda 311’ (−8.4 %). Our results indicated that high O3 exposure at grain filling stage would have greater negative impacts on the high yielding modern cultivar relative to the old one with lower yield.