Carbon assimilation by most ecosystems requires ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Its kinetic parameters are likely to have evolved in parallel with intracellular CO₂ availability, with the result that faster forms of Rubisco occur in species with CO₂-concentrating mechanisms. The Rubisco catalytic properties were determined and evaluated in relation to growth and carbon assimilation capacity in Mediterranean Limonium species, inhabiting severe stress environments. Significant kinetic differences between closely related species depended on two amino acid substitutions at functionally important residues 309 and 328 within the Rubisco large subunit. The Rubisco of species facing the largest CO₂ restrictions during drought had relatively high affinity for CO₂ (low Michaelis-Menten constant for CO₂ (K_c)) but low maximum rates of carboxylation (kcatc ), while the opposite was found for species that maintained higher CO₂ concentrations under similar conditions. Rubisco kinetic characteristics were correlated with photosynthetic rate in both well-watered and drought-stressed plants. Moreover, the drought-mediated decrease in plant biomass accumulation was consistently lower in species with higher Rubisco carboxylase catalytic efficiency (kcatc /K_c). The present study is the first demonstration of Rubisco adaptation during species diversification within closely related C₃ plants, revealing a direct relationship between Rubisco molecular evolution and the biomass accumulation of closely related species subjected to unfavourable conditions.