Interest in the thermal acclimation of photosynthesis has been stimulated by the increasing relevance of climate change. However, little is known about intra-specific variations in thermal acclimation and its potential for breeding. In this article, we examined the difference in thermal acclimation between alfalfa (Medicago sativa) cultivars originating from contrasting origins, and sought to analyze the mechanisms in play. A series of experiments was carried out at seven growth temperatures between 5 and 35 °C using four cultivars from temperate and Mediterranean origin. Leaf traits, the photosynthetic rate at 25 °C (A400 (25)), the photosynthetic rate at optimal temperature (A400 (opt)), the thermal optimum of photosynthesis (Topt), and the photosynthetic parameters from the Farqhuar model were determined. Irrespective of cultivar origin, a clear shift in the temperature responses of photosynthesis was observed as a function of growth temperature, affecting thermal optimum of photosynthesis, photosynthetic rate at optimal temperature and photosynthetic rate at 25 °C. For both cultivars, Topt values increased linearly in leaves grown between 5 and 35 °C. Relative homeostasis of A400 (25) and A400 (opt) was found between 10 °C and 30 °C growth temperatures, but sharp declines were recorded at 5 and 35 °C. This homeostasis was achieved in part through modifications to leaf nitrogen content, which increased at extreme temperatures. Significant changes were also recorded regarding nitrogen partitioning in the photosynthetic apparatus and in the temperature dependence of photosynthetic parameters. The cultivars differed only in terms of the temperature response of photosynthetic parameters, with Mediterranean genotypes displaying a greater sensitivity of the maximum rate of Rubisco carboxylation to elevated temperatures. It was concluded that intra-specific variations in the temperature acclimation of photosynthesis exist among alfalfa cultivars, but that Mediterranean genotypes presented no evidence of superior performance at high temperatures., (Published by Oxford University Press on behalf of the Annals of Botany Company.)