The ongoing global change could be an additional threat to the establishment and the long-term survival of Populus nigra L., an emblematic European riparian species. With the general aim of gaining insights into the adaptive potential of this species, we (i) quantified variations within and among three French P. nigra populations for key physiological attributes, i.e., water-use efficiency (assessed from bulk leaf carbon isotope discrimination, Δ13C), growth performance and related leaf traits, (ii) examined genotype and population by environment interactions, and (iii) explored the relationship between Δ13C and growth. Thirty genotypes were sampled in each of three naturally established populations and grown in two different sites, Orléans (ORL) and Guémené-Penfao (GMN). In ORL, two similar plots were established and different watering regimes were applied in order to test for the drought response. Significant variations were observed for all traits within and among populations irrespective of site and watering. Trait variation was larger within than among populations. The effect of drought was neither genotype- nor population-dependent, contrary to the effect of site. The population ranking was maintained in all sites and watering regimes for the two most complex traits: Δ13C and growth. Moreover, these two traits were unrelated, which indicates that (i) water-use efficiency and growth are largely uncoupled in this species, and (ii) the environmental factors driving genetic structuration for Δ13C and growth act independently. The large variations found within populations combined with the consistent differences among populations suggest a large adaptive potential for P. nigra.