Drought is expected to become an increasingly important factor limiting tree growth caused by climate change. Two divergent clones of Populus nigra (58-861 and Poli) originating from contrasting environments were subjected to water limitation (WL) to elucidate whether they differ in tolerance to drought, which mechanisms to avoid stress they exhibit and whether drought has an impact on the interactions between roots and shoots. Limiting water availability caused photosynthetic rate and total non-structural carbohydrate (TNC) levels to decrease in 58-861. However, starch-degrading enzyme activity and gene expression were induced in roots, and soluble sugar levels were higher than in well-watered (WW) plants. These data suggest that assimilation and partitioning of carbon to the roots are decreased, resulting in mobilization of stored starch. In contrast, the photosynthetic rate of Poli was reduced only late in the treatment, and carbohydrate levels in WL plants were higher than in WW plants. Superoxide dismutase (SOD) activity and gene expression were higher in Poli than in 58-861, even in WW plants, leading to a higher capacity to defend against oxidative stress.