- Land-use changes are one of the most important factors causing environmental transformations and species diversity alterations. The aim of the current study was to develop a geoinformatics-based framework to quantify alpha and beta diversity indices in two sites in Israel with different land-uses, i.e., an agricultural system of fruit orchards, an afforestation system of planted groves, and an unmanaged system of groves. The framework comprises four scaling steps: (1) classification of a tree species distribution (SD) map using imaging spectroscopy (IS) at a pixel size of 1 m; (2) estimation of local species richness by calculating the alpha diversity index for 30-m grid cells; (3) calculation of beta diversity for different land-use categories and sub-categories at different sizes; and (4) calculation of the beta diversity difference between the two sites. The SD was classified based on a hyperspectral image with 448 bands within the 380–2500 nm spectral range and a spatial resolution of 1 m. Twenty-three tree species were classified with high overall accuracy values of 82.57 and 86.93% for the two sites. Significantly high values of the alpha index characterize the unmanaged land-use, and the lowest values were calculated for the agricultural land-use. In addition, high values of alpha indices were found at the borders between the polygons related to the “edge-effect” phenomenon, whereas low alpha indices were found in areas with high invasion species rates. The beta index value, calculated for 58 polygons, was significantly lower in the agricultural land-use. The suggested framework of this study succeeded in quantifying land-use effects on tree species distribution, evenness, and richness. IS and spatial statistics techniques offer an opportunity to study woody plant species variation with a multiscale approach that is useful for managing land-use, especially under increasing environmental changes. This article is protected by copyright. All rights reserved.