13C discrimination between atmosphere and bulk leaf matter (Δ13Clb) is frequently used as a proxy for transpiration efficiency (TE). Nevertheless, its relevance is challenged due to: (1) potential deviations from the theoretical discrimination model, and (2) complex time integration and upscaling from leaf to whole plant. Six hybrid genotypes of Populus deltoides×nigra genotypes were grown in climate chambers and tested for whole-plant TE (i.e. accumulated biomass/water transpired). Net CO2 assimilation rates (A) and stomatal conductance (gs) were recorded in parallel to: (1) 13C in leaf bulk material (δ13Clb) and in soluble sugars (δ13Css) and (2) 18O in leaf water and bulk leaf material. Genotypic means of δ13Clb and δ13Css were tightly correlated. Discrimination between atmosphere and soluble sugars was correlated with daily intrinsic TE at leaf level (daily mean A/gs), and with whole-plant TE. Finally, gs was positively correlated to 18O enrichment of bulk matter or water of leaves at individual level, but not at genotype level. We conclude that Δ13Clb captures efficiently the genetic variability of whole-plant TE in poplar. Nevertheless, scaling from leaf level to whole-plant TE requires to take into account water losses and respiration independent of photosynthesis, which remain poorly documented.