With randomized complete block design, long-term water use efficiency (WUE_L), photosynthetic parameters (net photosynthetic rate Pn, transpiration rate Tr, stomatal conductance Gs, instantaneous water use efficiency WUE_i, maximum photosynthetic rate P max, light saturation pointLSP, carboxylic efficiency Ce), stomatal properties (frequency, size, superior/inferior stoma amount ratio) and root/shoot ratio of twelve Populus deltoids clones were studied under water stress in a greenhouse. Four water treatments were designed, namely, well-watered condition, slight water stress, moderate water stress, and severe water stress. Although the volumes of irrigated water for different treatments were the same, the irrigation intervals were strictly controlled. The results showed that long-term water use efficiency (WUE_L) differences among tested clones were obvious and became greater with the increase in water stress. J₂, J₆, J₇, J₈, and J₉ were excellent clones with high WUE_L. Stomatal frequency, stomatal size, Gs, Tr, Pn, and root/shoot ratio of J₂et al were moderate, whereas the superior/inferior ratio, P_max, LSP, Ce of J2et al were higher than other clones. The interrelations between WUE_L and the physiological parameters were analyzed. The results revealed that the WUE_L differences were induced by a series of physiological parameters, and clones with higher WUE_L always had strong photosynthetic capacity, higher WUE_i, and optimum root/shoot ratio.