• Water use efficiency (WUE), oftentimes estimated as transpiration efficiency (TE): the amount of biomass produced with regard to the water used, has not yet been used as a breeding trait to select poplar genotypes with simultaneously high productivity and conservation of water. Before its application as a selection target, evidence must be presented showing that WUE or its estimators remain constant with age and across environmental conditions. • We conducted a rainfall exclusion experiment in the field on two Populus euramericana (Moench.) and two Populus nigra (L.) genotypes, and assessed leaf-level (A/g s) and whole-plant WUE (DMT/WU as well as their components and related traits. Then, we aimed to compare these results with the same poplar genotypes grown in a glasshouse under contrasting water availability. • Despite a reduction of soil water content and whole-plant transpiration, growth was stimulated in the rainfall exclusion plot, likely as a result of an increased nitrogen assimilation. However, TE values between the glasshouse and the field were similar, and genotype ranking remained fairly constant for transpiration, carbon isotopic discrimination (∆, as a proxy for Wi) and TE. Moreover, even though the drivers of WUE in both experiments were different, increases of WUE measured as ∆ or TE was not associated with lower biomass production. Relatively good agreement was found between ∆ and TE in the field, absence of a similar correlation in the glasshouse is discussed. • These results suggest that ∆ may be a good proxy for TE, and could be used, both as a breeding target for genotype selection in glasshouses without impacting biomass production when planted in the field. However, reduced water availability modified the genotype ranking more significantly than between the field/glasshouse experiments, suggesting a diversity of poplar response to drought that should be considered in breeding strategies. ABBREVIATIONS WUE water use efficiency Wi leaf intrinsic water use efficiency measured by leaf gas exchange (A/g s) TE whole-plant transpiration efficiency (biomass production over water used, DM T /WU) ∆ carbon isotope discrimination δ carbon isotope composition A net CO 2 assimilation C i internal CO 2 concentration g s stomatal conductance to water vapor WU whole plant water use E daily whole-plant transpiration per unit area VPD vapor pressure deficit Vc max maximum CO 2 carboxylation rate J max maximum photosynthetic electron flux A max net CO 2 assimilation measured under saturating atmospheric CO 2 concentration SWC soil water content Ψ p predawn leaf water potential H tree height https://doi.