We have characterized poplar aquaporins (AQPs) to investigate their possible functions in differential drought responses of Populus balsamifera and Populus simonii×balsamifera leaves. Plants were exposed to mild and severe levels of drought stress and to drought stress recovery treatment, and their responses were compared with well-watered controls. Compared with P. balsamifera, P. simonii×balsamifera used drought avoidance as the main drought resistance strategy, and rapidly reduced stomatal conductance in response to stress. This strategy is correlated with growth rate reductions. Eleven AQPs were transcriptionally profiled in leaves from these experiments and five were functionally characterized for water channel activity. PIP1;3 and PIP2;5 were among the most highly expressed leaf AQPs that were responsive to drought. Expression of PIP1;3 and five other AQPs increased in response to drought in the leaves of P. simonii×balsamifera but not in P. balsamifera, suggesting a possible role of these AQPs in water redistribution in the leaf tissues. PIP2;5 was upregulated in P. balsamifera, but not in P. simonii×balsamifera, suggesting that this AQP supports the transpiration-driven water flow. Functional characterization of five drought-responsive plasma membrane intrinsic proteins (PIPs) demonstrated that three PIP2 AQPs (PIP2;2, PIP2;5, PIP2;7) functioned as water transporters in Xenopus laevis oocytes, while the two PIP1 AQPs (PIP1;2 and PIP1;3) did not, consistent with the notion that they may be functional only as heterotetramers.