13C discrimination in organic matter with respect to atmospheric CO2 (D13C) is under tight genetic control in many plant species, including the pedunculate oak (Quercus robur L.) full-sib progeny used in this study. D13C is expected to reflect intrinsic water use efficiency, but this assumption requires confirmation due to potential interferences with mesophyll conductance to CO2, or post-photosynthetic discrimination. In order to dissect the observed D13C variability in this progeny, six genotypes that have previously been found to display extreme phenotypic values of D13C [either very high (‘high D’) or low (‘low D’) phenotype] were selected, and transpiration efficiency (TE; accumulated biomass/transpired water), net CO2 assimilation rate (A), stomatal conductance for water vapour (gs), and intrinsic water use efficiency (Wi¼A/gs) were compared with D13C in bulk leaf matter, wood, and cellulose in wood. As expected, ‘high D’ displayed higher values of D13C not only in bulk leaf matter, but also in wood and cellulose. This confirmed the stability of the genotypic differences in D13C recorded earlier. ‘High D’ also displayed lower TE, lower Wi, and higher gs. A small difference was detected in photosynthetic capacity but none in mesophyll conductance to CO2. ‘High D’ and ‘low D’ displayed very similar leaf anatomy, except for higher stomatal density in ‘high D’. Finally, diurnal courses of leaf gas exchange revealed a higher gs in ‘high D’ in the morning than in the afternoon when the difference decreased. The gene ERECTA, involved in the control of water use efficiency, leaf differentiation, and stomatal density, displayed higher expression levels in ‘low D’. In this progeny, the variability of D13C correlated closely with that of Wi and TE. Genetic differences of D13C and Wi can be ascribed to differences in stomatal conductance and stomatal density but not in photosynthetic capacity.