Due to global warming, a significant increase in frequency and severity of drought episodes is predicted in the Mediterranean Region. Olive (Olea europaea L.) is a relatively drought-tolerant tree species cultivated in Mediterranean region. However, prolonged hot and dry summer can induce hydraulic stress, leading to fruit yield reduction. Hence, studies about the adaptation and tolerance of olive trees to stress are fundamental to better understand how different cultivars will cope with ongoing climate change. Mature trees of two high-quality cultivars (Moraiolo and Maurino), growing in Central Italy, were selected to evaluate differences in growth, fruit production, and tolerance to hot and dry conditions. To study Intra and inter-annual physiological responses (stomatal sensitivity, photosynthetic capacity and intrinsic water-use efficiency) to local climate, carbon and oxygen stable isotopes were investigated in tree rings of two olive cultivars during three growing seasons of 2020, 2021 and 2022. The two cultivars differed in seasonal ?13C trends during the three years. Tree ring ?13C values increased during earlywood formation and decreased in the latewood. The increasing trend in ?13C, from early spring to summer indicates a decrease in photosynthetic isotopic discrimination. This would imply an increase in intrinsic water-use efficiency in response to increasing temperature and decreasing water availability. In this regard, cv. Maurino showed a more limited range of variation in comparison to cv. Moraiolo. This may be related to different stomatal sensitivity, leading to more limited stress tolerance in Maurino with respect to Moraiolo. In all the years, Moraiolo reached significantly lower ?13C values in the winter and higher ?13C values in the summer, indicating a higher stomatal responsiveness and a higher phenotypic plasticity in response to changes in environmental conditions during the vegetative and reproductive season. In agreement with this hypothesis, the ?18O values of the 3-year-old branches were significantly higher in trees from Maurino, suggesting higher transpiration rates. Hence, Moraiolo cultivar has the potential to better tolerate thermal variations and drought conditions in response to Mediterranean climate change.