The efficiency of water use to produce biomass is a key trait in designing sustainable bioenergy-devoted systems. We characterized variations in the carbon isotope composition (δ13C) of leaves, current year wood and holocellulose (as proxies for water use efficiency, WUE) among six poplar genotypes in a short-rotation plantation. Values of δ13Cwood and δ13Cholocellulose were tightly and positively correlated, but the offset varied significantly among genotypes (0.79–1.01‰). Leaf phenology was strongly correlated with δ13C, and genotypes with a longer growing season showed a higher WUE. In contrast, traits related to growth and carbon uptake were poorly linked to δ13C. Trees growing on former pasture with higher N-availability displayed higher δ13C as compared with trees growing on former cropland. The positive relationships between δ13Cleaf and leaf N suggested that spatial variations in WUE over the plantation were mainly driven by an N-related effect on photosynthetic capacities. The very coherent genotype ranking obtained with δ13C in the different tree compartments has some practical outreach. Because WUE remains largely uncoupled from growth in poplar plantations, there is potential to identify genotypes with satisfactory growth and higher WUE.