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Accurate prediction of food crop yield is of great significance for global food security and regional trade stability. Since remote sensing data collected from unmanned aerial vehicle (UAV) platforms have the features of flexibility and high resolution, these data can be used as samples to develop regional regression models for accurate prediction of crop yield at a field scale. The primary objective of this study was to construct regional prediction models for winter wheat yield based on multi-spectral UAV data and machine learning methods. Six machine learning methods including Gaussian process regression (GPR), support vector machine regression (SVR) and random forest regression (RFR) were used for the construction of the yield prediction models. Ten vegetation indices (VIs) extracted from canopy spectral images of winter wheat acquired from a multi-spectral UAV at five key growth stages in Xuzhou City, Jiangsu Province, China in 2021 were selected as the variables of the models. In addition, in situ measurements of wheat yield were obtained in a destructive sampling manner for prediction algorithm modeling and validation. Prediction results of single growth stages showed that the optimal model was GPR constructed from extremely strong correlated VIs (ESCVIs) at the filling stage (R2 = 0.87, RMSE = 49.22 g/m2, MAE = 42.74 g/m2). The results of multiple stages showed GPR achieved the highest accuracy (R2 = 0.88, RMSE = 49.18 g/m2, MAE = 42.57 g/m2) when the ESCVIs of the flowering and filling stages were used. Larger sampling plots were adopted to verify the accuracy of yield prediction; the results indicated that the GPR model has strong adaptability at different scales. These findings suggest that using machine learning methods and multi-spectral UAV data can accurately predict crop yield at the field scale and deliver a valuable application reference for farm-scale field crop management. [ABSTRACT FROM AUTHOR]