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Leaf biochemical properties have been widely assessed using hyperspectral reflectance information by inversion of PROSPECT model or by using hyperspectral indices, but few studies have focused on arid ecosystems. As a dominant species of riparian ecosystems in arid lands, Populus euphratica Oliv. is an unusual tree species with polymorphic leaves along the vertical profile of canopy corresponding to different growth stages. In this study, we evaluated both the inversed PROSPECT model and hyperspectral indices for estimating biochemical properties of P. euphratica leaves. Both the shapes and biochemical properties of P. euphratica leaves were found to change with the heights from ground surface. The results indicated that the model inversion calibrated for each leaf shape performed much better than the model calibrated for all leaf shapes, and also better than hyperspectral indices. Similar results were obtained for estimations of equivalent water thickness (EWT) and leaf mass per area (LMA). Hyperspectral indices identified in this study for estimating these leaf properties had root mean square error (RMSE) and R-2 values between those obtained with the two calibration strategies using the inversed PROSPECT model. Hence, the inversed PROSPECT model can be applied to estimate leaf biochemical properties in arid ecosystems, but the calibration to the model requires special attention.