The production and quality of grapes are determined by the hydric and nutritional status of the vine. In modern viticulture, the interface between grapevine and soil is represented by the rootstock, which modulates the uptake of water and nutrients. Thus, selection of new rootstocks for abiotic stress tolerance represents an adaptation strategy for viticulture to the new environmental conditions imposed by climate change. The aim of this work is to evaluate the water-use efficiency (WUE) and the nutritional status of a selection of 30 new rootstock genotypes, originating from several breeding programs. WUE and gas exchange were measured for each genotype in two environments characterized by different water availability, and the levels of N, P, K, Mg, Ca, Na, Fe, Mn, and Zn in the leaf blades were determined during the phenological stages of flowering and ripening. Water availability was different in the two environments, affecting gas exchange, with mean values of carbon assimilation of 10.43 ± 0.32 and 3.84 ± 0.34 µmol CO2 m−2 s−1, respectively. Genotypes were classified according to their water stress tolerance, vigor, and affinity for macronutrients and micronutrients. A total of 14 genotypes showed tolerance to water stress, and 10 of them reported high vigor and carbon assimilation. Furthermore, the majority of water-stress-tolerant genotypes had greater affinity for Mg than for K. In further studies, the most promising rootstocks will be evaluated in grafting combination with Vitis vinifera.