To determine the effect of CO2 laser pretreatment of wheat seeds on the physiological tolerance of seedlings to chilling stress, wheat seeds were exposed to CO2 laser radiation for 300 s. After being cultivated for 48 h at 25°C, the wheat seedlings were subjected to chilling stress for 24 h. Selected physiological and biochemical parameters were measured in 6-day-old seedlings. We observed that chilling stress enhanced the concentrations of malondialdehyde and oxidized glutathione while decreasing the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione in the wheat leaves compared with controls. When the chilling stress was preceded by CO2 laser irradiation, the concentrations of malondialdehyde and oxidized glutathione were decreased while the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione increased. Furthermore, chilling stress decreased the biomass, biophoton intensity and GHS/GSSG ratios of seedlings while these parameters increased when the seedlings were treated with CO2 laser irradiation prior to the chilling stress. The results suggest that a suitable dose of CO2 laser stimulation can enhance the physiological tolerance of wheat seedlings to chilling stress.