Cells of three strains of Listeria monocytogenes, one a reference strain ATCC 19115 and two strains isolated from meat, were cold shocked at refrigeration (+4C) and freezing (−20C) temperatures during one night. Then, their fatty acids were extracted and their composition identified by gas chromatography coupled with mass spectrometry. The results showed that low thermal adaptation response of L. monocytogenes ATCC 19115 was different from that of the two recent food isolates L. monocytogenes. The three experimented strains showed a decrease of anteiso-C17:0 and an increase of anteiso-C15:0 rates. In addition, after freezing, the cellular fatty acids were detected as a signature of the membrane changes that give rise to such authenticity. The structural modification seems to be an adaptation form, allowing intracellular ice crystallization enhancement during freezing and water mobility during storage. These results demonstrated that the L. monocytogenes studied strains showed various behaviors regarding low-temperature stress maintaining membrane fluidity.