Genetic diversity in intrinsic water use efficiency (Wi) has been studied within a pedunculate oak (Quercus robur L.) family. Wi is defined at the leaf level as the ratio between net CO2 assimilation rate and stomatal conductance to water vapour. For large scale phenotyping, this trait can be estimated by measuring carbon isotope composition (δ13C) on organic material (leaves, wood). Genetic determinism for Wi was evaluated using a genetic mapping/quantitative trait loci (QTLs) approach and several regions were detected on the genetic map. Phenotypes of this family have been investigated intensively and partitioning of the observed diversity of Wi has shown a strong influence of stomatal conductance, whereby variation in Wi was related to variation in stomatal density and stomatal sensitivity to diurnal courses of irradiance. A modelling approach was used to detect more details of functional differences among genotypes. A complementary molecular biology approach was used to create a list of possible candidate genes related to the variability of Wi, using differential gene expression, as well as the study of the ERECTA gene, which is known to be related to variation in Wi in Arabidopsis thaliana.