Global warming is expected to have inexorable and profound effects on marine ecosystems, particularly in foundation species such as seagrasses. Identifying responses to warming and comparing populations across natural temperature gradients can inform how future warming will impact the structure and function of ecosystems. Here, we investigated how thermal environment, intra-shoot and spatial variability modulate biochemical responses of the Mediterranean seagrassPosidonia oceanica. Through a space-for-time substitution study, Fatty acid (FA) profiles on the second and fifth leaf of the shoots were quantified at eight sites in Sardinia along a natural sea surface temperature (SST) summer gradient (about 4 °C). Higher mean SST were related to a decrease in the leaf total fatty acid content (LTFA), a reduction in polyunsaturated fatty acids (PUFA), omega-3/omega-6 PUFA and PUFA/saturated fatty acids (SFA) ratios and an increase in SFA, monounsaturated fatty acids and carbon elongation index (CEI, C18:2 n-6/C16:2 n-6) ratio. Results also revealed that FA profiles were strongly influenced by leaf age, independently of SST and spatial variability within sites. Overall, this study evidenced that the sensitive response ofP. oceanicaFA profiles to intra-shoot and spatial variability must not be overlooked when considering their response to temperature changes.