Most of the vineyards around the world are in areas characterized by seasonal drought, where water deficits and high temperatures represent severe constraints on the regular grapevine growth cycle. Although grapevines are well adapted to arid and semi-arid environments, water stress can cause physiological changes, from mild to irreversible. Screening of available Vitis spp. genetic diversity for new rootstock breeding programs has been proposed as a way for which new viticulture challenges may be faced. In 2014, novel genotypes (M-rootstocks) were released from the University of Milan. In this work, the behavior of M1, M3 and M4 in response to decreasing water availabilities (80%, 50% and 20% soil water content, SWC) was investigated at the physiological and gene expression levels, evaluating gas exchange, stem water potential and transcript abundances of key genes related to ABA (abscisic acid) biosynthesis (VvZEP, VvNCED1 and VvNCED2) and signaling (VvPP2C4, VvSnRK2.6 and VvABF2), and comparing them to those of cuttings of nine commercial rootstocks widely used in viticulture. M-rootstocks showed a change at physiological levels in severe water-stressed conditions (20% soil water content, SWC), reducing the stomatal conductance and stem water potential, but maintaining high photosynthetic activity. Water use efficiency was high in water-limiting conditions. The transcriptional changes were observed at 50% SWC, with an increment of transcripts of VvNCED1 and VvNCED2 genes. M-rootstocks showed similar behavior to 1103P and 110R rootstocks, two highly tolerant commercial genotypes. These rootstocks adopted a tolerant strategy to face water-stressed conditions.