Abstract: In order to evaluate the cold resistance of mutagenic lines of perennial ryegrass and obtain more cold resistance perennial ryegrasses, we studied the physiological and biochemical responses of perennial ryegrasses under low temperature (above zero) treatment, and the materials of this experiment are five mutagenic lines-G3-2, G10-5, G16-1, CK9-3, CK14-3 and one variety with good cold resistance-‘Pinnacle’. The results of this study show that, in the process of cold acclimation, the leaf water content decrease: G16-1’s leaf water content decreases by up to 3.12%. Relative membrane permeability increases and malondialdehyde accumulates: CK14-3 has the lowest relative membrane permeability, which is 25.40%; while CK9-3 accumulates the lowest malondialdehyde content, which is 38.06 μmol/g. The osmotic regulation substances increase: CK14-3 and ‘Pinnacle’ have lower proline content with 0.18% and 0.15% respectively; G16-1’s solute protein content increases by 83.08%, which is the largest; ‘Pinnacle’s solute sugar content is the lowest increaseing by 34.18%, and CK9-3 takes the second place with 53.41%; the reducing sugar content of G10-5 and G16-1 weigh against ‘Pinnacle’ with 9.01% and 9.79% separately. The antioxidant enzymes are enhanced, the superoxide dismutase activity and catalase activity of CK14-3 are lower, with5171.21 U/(g·h) and 171.04 U/(g·min) respectively, but the changing rate are quicker with 169.07% and 47.06% respectively. As to potential photochemical efficiency, G3-2 and G16-1 decreases by 3.46% and 3.44% separately, which are small. By principal component analysis and membership function calculating, the cold-resistance ranking of the experiment materials is ‘Pinnacle’, G16-1, CK9-3, G10-5, CK14-3, G3-2 from strong to weak. The experiment materials are classified into three types: ‘Pinnacle’’s cold-tolerance is strong, G16-1, CK9-3, G10-5 and CK14-3’s are medium and G3-2’s is poor. Considering the results, we find that the more cold-resistance the perennial ryegrass is, the less relative membrane permeability it has; the less leaf water content, malondialdehyde content, proline content and reducing sugar content it have, the quicker the soluble protein content, superoxide dismutase and catalase activities change, the slower solute sugar content and potential photochemical efficiency change.