Water deficit presents new challenges for fruit production, among which, obtaining and selecting tolerant genotypes has become relevant. The aim of this work was to characterize novel Prunus rootstocks in regard to tolerance to water deficit as a key trait to cope the challenges for agriculture imposed by the climate change expected in the near future. Here, we report the assessment of physiological parameters and growth responses to water deficit of four interspecific Prunus rootstock hybrids. Two commercially available rootstocks, ROOTPAC (R) 40 and ROOTPAC (R) 20, and two experimental genotypes, AGAF 0301-07 and ADAGAF 0403 were evaluated. Plants were subjected to two irrigation treatments (well-watered and water deficit) during 33 days. During this period, stomatal conductance (g(s)) and photosynthetic CO2 assimilation (P-n) were measured. At the end of the experiment, biomass accumulation was registered for all the genotypes, while the expression of seven aquaporins belonging to plasma membrane intrinsic proteins subfamily (PIPs) was assessed in ROOTPAC (R) 40 and ROOTPAC (R) 20. ROOTPAC (R) 20 had a decrease in the aquaporin gene expression in roots and this could be associated with its early decline in transpiration. Biomass accumulation was lower in ROOTPAC (R) 20, AGAF 0301-07 and ADAGAF 0403 due to the water deficit. However, ROOTPAC (R) 40 showed a larger whole-plant water use efficiency under water deficit because of a later reduction in relative transpiration under low fractions of transpirable soil water. This response resulted in higher biomass accumulation in ROOTPAC (R) 40 than the other genotypes. We identified ROOTPAC (R) 40 as a tolerant genotype to water deficit, which is majorly explained by a later reduction in relative transpiration under water deficit.