Vegetation diversity is a crucial indicator for evaluating grassland ecosystems. Remote sensing technology has great potential in assessing grassland vegetation diversity. In this study, the relationship between remote sensing indices and species diversity was investigated at varying spatial and temporal scales in Bayanbulak Grassland National Nature Reserve, China. Spectral variation, defined as the coefficient of variation in vegetation indices, was used as a proxy for species diversity, which was quantified using species diversity indices. The “spectral diversity-species diversity” relationship was validated across diverse spatial scales and between different years using Sentinel-2 images and ground investigation data. This study found that Kendall’s τ coefficients showed the best performance in evaluating the relationship between the coefficient of variation in VIs (CVVIs) and species diversity index. The highest τ value was observed for CVNDVI in 2017 (τ = 0.660, p < 0.01), followed by the Shannon index in 2018 (τ = 0.451, p < 0.01). In addition, CVEVI demonstrated a significant positive correlation with the Shannon-Wiener Index at the 50 m scale (τ = 0.542), and the highest relationship τ between CVNDVI and the Shannon-Wiener Index was observed at the 100 m scale (τ = 0.660). The Shannon-Wiener Index in relation to CVVIs performs better in representing changes in grassland vegetation. Spatial scales and vegetation indices influence the assessment of grassland vegetation diversity. These findings underscore the critical role of remote sensing technology in assessing grassland vegetation diversity across various scales, offering valuable support tools for measuring regional grassland vegetation diversity.
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