The accurate estimation of nitrogen content in crop plants is the basis of precise nitrogen fertilizer management. Unmanned aerial vehicle (UAV) imaging technology has been widely used to rapidly estimate the nitrogen in crop plants, but the accuracy will still be affected by the variety, the growth stage, and other factors. We aimed to (1) analyze the correlation between the plant nitrogen content of winter wheat and spectral, texture, and structural information; (2) compare the accuracy of nitrogen estimation at single versus multiple growth stages; (3) assess the consistency of UAV multispectral images in estimating nitrogen content across different wheat varieties; (4) identify the best model for estimating plant nitrogen content (PNC) by comparing five machine learning algorithms. The results indicated that for the estimation of PNC across all varieties and growth stages, the random forest regression (RFR) model performed best among the five models, obtaining R2, RMSE, MAE, and MAPE values of 0.90, 0.10%, 0.08, and 0.06%, respectively. Additionally, the RFR estimation model achieved commendable accuracy in estimating PNC in three different varieties, with R2 values of 0.91, 0.93, and 0.72. For the dataset of the single growth stage, Gaussian process regression (GPR) performed best among the five regression models, with R2 values ranging from 0.66 to 0.81. Due to the varying nitrogen sensitivities, the accuracy of UAV multispectral nitrogen estimation was also different among the three varieties. Among the three varieties, the estimation accuracy of SL02-1 PNC was the worst. This study is helpful for the rapid diagnosis of crop nitrogen nutrition through UAV multispectral imaging technology.