Global warming is emerging as one of the most critical threats to terrestrial and marine species worldwide. This study assessed the effects of simulated warming events in culture on two seagrass species, Posidonia oceanica and Cymodocea nodosa, which play a key role in coastal ecosystems of the Mediterranean Sea. Changes in fatty acids as key metabolic indicators were assessed in specimens from two geographical populations of each species adapted to different in situ temperature regimes. Total fatty acid (TFA) content and composition were compared in C. nodosa and P. oceanica from natural populations and following exposure to heat stress in culture. After heat exposure, individuals of C. nodosa and P. oceanica adapted to colder temperatures in situ accumulated significantly more TFA than controls. For both species, the proportion of polyunsaturated fatty acids (PUFA) decreased, and the percentage of saturated fatty acids (SFA) increased significantly after the heat treatment. These results highlight that populations of both species living at warmest temperatures in situ were more thermo-tolerant and exhibited a greater capacity to cope with heat stress by readjusting their lipid composition faster. Finally, exposure of seagrasses to warmer conditions may induce a decrease in PUFA/SFA ratio which could negatively affect their nutritional value and generate important consequences in the healthy state of next trophic levels.