Poplar genotypes differ in transpiration efficiency (TE) at leaf and whole-plant level under similar conditions. We tested whether atmospheric vapour pressure deficit (VPD) affected TE to the same extent across genotypes. Six Populus nigra genotypes were grown under two VPD. We recorded (1) C-13 content in soluble sugars; (2) O-18 enrichment in leaf water; (3) leaf-level gas exchange; and (4) whole-plant biomass accumulation and water use. Whole-plant and intrinsic leaf TE and C-13 content in soluble sugars differed significantly among genotypes. Stomatal conductance contributed more to these differences than net CO2 assimilation rate. VPD increased water use and reduced whole-plant TE. It increased intrinsic leaf-level TE due to a decline in stomatal conductance. It also promoted higher O-18 enrichment in leaf water. VPD had no genotype-specific effect. We detected a deviation in the relationship between C-13 in leaf sugars and C-13 predicted from gas exchange and the standard discrimination model. This may be partly due to genotypic differences in mesophyll conductance, and to its lack of sensitivity to VPD. Leaf-level C-13 discrimination was a powerful predictor of the genetic variability of whole-plant TE irrespective of VPD during growth.