Bermudagrass (Cynodon dactylon) is a typical and widely used warm-season turfgrass. Low temperature is one of the key environmental stress limiting its utility. However, little information is available about the differences of cold response between bermudagrass genotypes. Here, we analyzed antioxidant defense system and fatty acid composition in cold-resistant genotype WBD128 and cold-sensitive genotype WBDg17 exposed to chilling stress. Low temperature (4 °C) significantly decreased the relative water content, whereas increased the H2O2 and O2 − contents, more profoundly for WBDg17. Under chilling condition, WBD128 had higher anti O2 − activity than WBDg17. Besides, the contents of total glutathione, reduced glutathione (GSH) and its oxidized form (GSSG) were markedly increased by low temperature in both genotypes, whereas WBD128 had significantly higher values of GSH, total glutathione, and GSH/GSSG ratio than WBDg17. Moreover, chilling stress increased saturated fatty acids (SFAs) percentage (palmitic acid and stearic acid) in WBDg17. After chilling treatment, the proportion of linoleic acid decreased in both genotypes, particularly in WBDg17. As for unsaturated fatty acids (UFAs), the percentage of linolenic acid was increased in WBD128. In addition, chilling treatment decreased the values of double bond index (DBI), UFA/SFA ratio as well as degree of unsaturation in WBDg17. Finally, chilling stress altered the expression patterns of the genes, which encode one kind of late embryogenesis abundant proteins (LEA), superoxide dismutase (Cu/Zn SOD) C-repeat-binding factor/DRE-binding factor (CBF1), and peroxidase (POD-2). Collectively, our results revealed that natural variation of chilling tolerance in bermudagrass genotypes may be largely associated with the alterations of antioxidant defense system and fatty acid composition.