Low temperature is a major factor limiting the productivity and geographical distribution of many plant species. In this study, we investigated the effect of chilling stress (10 o C) on seedling growth in two sweet sorghum (Sorghum bicolor (L.) Moench) inbred lines (M-81E and Roma). Results showed that the chilling resistance of M-81E was higher than that of Roma. The Fv/Fm in leaves of M-81E decreased less than that of Roma during chilling stress. After 24 h of chilling stress, the Fv/Fm of M-81E and Roma decreased by 24.3 and 45.8%, respectively. Fo was also affected significantly during chilling stress. Malondialdehyde (MDA), an indicator of lipid peroxidation caused by ROS, increased during chilling stress. The contents of MDA increased less in leaves of M-81E than that in Roma under chilling stress. The antioxidant enzymes (SOD and APX) activity of M-81E was higher than those of Roma during chilling stress. The unsaturated fatty acid content and the double bond index (DBI) of major membrane lipids of MGDG, DGDG, SQDG, PC, PE and PG of M-81E significantly increased after 24 h of chilling treatment (10 o C). The DBI of MGDG, DGDG, SQDG, PC and PG of Roma significantly decreased. These results showed that the chilling tolerance of M-81E was higher than that of Roma by increasing of unsaturated fatty acid in membrane lipid and powerful protective enzyme system at seedling stage.