Photosynthetic carbon assimilation and transpirational water loss play an important role in the yield and the carbon sequestration potential of bioenergy-devoted cultures of fast-growing trees. For six poplar (Populus) genotypes in a short-rotation plantation, we observed significant seasonal and genotypic variation in photosynthetic parameters, intrinsic water-use efficiency (WUEi) and leaf stable isotope composition (δ13C and δ18O). The poplars maintained high photosynthetic rates (between 17.8 and 26.9 μmol m−2 s−1 depending on genotypes) until late in the season, in line with their fast-growth habit. Seasonal fluctuations were mainly explained by variations in soil water availability and by stomatal limitation upon photosynthesis. Stomatal rather than biochemical limitation was confirmed by the constant intrinsic photosynthetic capacity (Vcmax) during the growing season, closely related to leaf nitrogen (N) content. Intrinsic water-use efficiency scaled negatively with carbon isotope discrimination (Δ13Cbl) and positively with the ratio between mesophyll diffusion conductance (gm) and stomatal conductance. The WUEi – Δ13Cbl relationship was partly influenced by gm. There was a trade-off between WUEi and photosynthetic N-use efficiency, but only when soil water availability was limiting. Our results suggest that seasonal fluctuations in relation to soil water availability should be accounted for in future modelling studies assessing the carbon sequestration potential and the water-use efficiency of woody energy crops.