Photosynthesis and antioxidant defense system were investigated under chilling stress without (Ch, 25-4 °C) and with acclimation (AcCh, 14-4 °C) in winter (Sabalan) and spring (Zagros) wheat (Triticum aestivum L.) cultivars. Maximum quantum efficiency of photosystem II and CO2 assimilation rate decreased in AcCh ‘Zagros’ but not in ‘Sabalan’, and in contrast, an increase in non-photochemical quenching was observed in ‘Sabalan’ but not in ‘Zagros’. Reduction of leaf starch content was observed in both cultivars while total soluble carbohydrates increased only in ‘Sabalan’ under both Ch and AcCh treatments. Activity of superoxide dismutase was significantly higher in Ch plants and activity of ascorbate peroxidase and catalase was slightly higher in Ch and AcCh plants of both cultivars compared with control. Activity of peroxidase increased in Ch and AcCh plants of ‘Zagros’ while phenylalanine ammoialyase (PAL) activity increased in AcCh ‘Sabalan’. Increase in the leaf content of H2O2 and malondialdehyde (MDA) was more prominent in ‘Zagros’ than in 'Sabalan'. According to our results, chilling tolerance in winter cultivar was associated with greater thermal dissipation, higher soluble carbohydrates content, greater PAL activity and lower H2O2 and MDA content. Furthermore, acclimated plants were not more protected against chilling injury compared with non-acclimated ones.