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Vegetation indices are frequently used for the non-destructive assessment of leaf chemistry, especially chlorophyll content. However, most vegetation indices were developed based on the statistical relationship between the spectral reflectance of the adaxial leaf surface and chlorophyll content, even though abaxial leaf surfaces may influence reflectance spectra because of canopy structure or the inclination of leaves. In the present study, reflectance spectra from both adaxial and abaxial leaf surfaces of Populus alba and Ulmus pumila var. pendula were measured. The results showed that structural differences of the two leaf surfaces may result in differences in reflectance and hyperspectral vegetation indices. Among 30 vegetation indices tested, R672/(R550 × R708) had the smallest difference (4.66% for P. alba, 2.30% for U. pumila var. pendula) between the two blade surfaces of the same leaf in both species. However, linear regression analysis showed that several vegetation indices (R850 − R710)/(R850 − R680), VOG2, D730, and D740, had high coefficients of determination (R2 > 0.8) and varied little between the two leaf surfaces of the plants we sampled. This demonstrated that these four vegetation indices had relatively stable accuracy for estimating leaf chlorophyll content. The coefficients of determination (R2) for the calibration of P. alba leaves were 0.92, 0.98, 0.93, and 0.95 on the adaxial surfaces, and 0.88, 0.87, 0.88, and 0.92 on the abaxial surfaces. The coefficients of determination (R2) for the calibration of U. pumila var. pendula leaves were 0.85, 0.91, 0.86, and 0.90 on adaxial surface, and 0.80, 0.80, 0.84, and 0.88 on abaxial surface. These four vegetation indices were readily available and were little influenced by the differences in the two leaf surfaces during the estimation of leaf chlorophyll content. [ABSTRACT FROM AUTHOR]