Mediterranean viticulture is highly dependent on soil water availability. Moreover, global warming is likely to increase average temperatures and the frequency of heat waves, thus leading to greater water scarcity. To cope with this situation, much research focused on the selection of genotypes with higher water use efficiency (WUE). Several previous studies indicated that WUE varies between genotypes of the Tempranillo cultivar with some showing interesting improvement in the leaf WUE. Here, we assessed the associations between the leaf WUE and physiological parameters in six selected Tempranillo clones. The plants were cultivated in pots and two water conditions were applied sequentially: a well-watered period followed by a moderate water stress and recovery phase. Growth parameters and intrinsic WUE were monitored during both periods. Pressure–volume and AN/Ci curves were measured after the plants were re-watered. At the end of the season, biomass accumulation and total irrigation water were used to calculate whole plant WUE (WUEWP). AN/Ci curves revealed significant differences in several photosynthetic parameters, including mesophyll conductance (gm), maximum velocity of carboxylation (Vcmax) and leaf respiration (R). Clones RJ51 and 1048 both exhibited high WUE, but presented distinct physiological traits: RJ51 had the highest gm, while genotype 1048, had the lowest R. This study confirms that physiological traits generally explain the intracultivar diversity in WUE observed in previous studies.