‘Midiron’ and ‘U3’ bermudagrass were exposed to conditions known to induce cold acclimation [e.g., 8/4°C (day/night) temperature, 10‐h photoperiod, 250 μmol m−2 s−1 photosynthetic photon flux density (PPFD)]. Tissues (leaves, crowns, and roots) were harvested stored frozen until processed. Polar membrane ids were isolated by thin layer chromatography, and fatty acids (FA) were separated and quantified by gas chromatography. Different organs from the same plant responded differentially to low temperature. Crowns showed the most dramatic changes in total FA content and composition and were the focus of this study. Overall, greater than 95% of the total FA content was accounted for by four FA species: palmitic acid (16:0), stearic acid (18:0), linoleic acid (18:2), and linolenic (18:3). Midiron (relatively cold‐tolerant) responded more rapidly to a greater extent than did U3 (relatively cold‐sensitive) as illustrated by the nearly four‐fold increase of unsaturated FA:saturated FA ratio for Midiron vs. U3, and by the significant difference between the double bond index of the two genotypes. These data suggest that specific desaturase enzymes (e.g., ω‐3 and ω‐6) are of fundamental importance in controlling membrane lipid/fatty acid composition in response to low temperature and ultimately in avoiding the winter damage suffered by bermudagrass along its northern boundary of adaptation.