Crop nitrogen status is a key indicator for evaluating crop growth, increasing yield and improving grain quality. Non-destructive and rapid assessment of leaf nitrogen is required for improving nitrogen management in wheat production. This study aims at identification of the quantitative relationship between leaf nitrogen accumulation and canopy reflectance spectra in winter wheat (Triticum aestivum L.) using three field experiments with different wheat varieties and nitrogen levels. Results showed that leaf nitrogen accumulation in wheat increased with increasing nitrogen rates. Canopy reflectance changed with increasing leaf nitrogen accumulation. Sensitivity bands occurred mainly during visible light and near infra-red, and strong correlation existed between red light and leaf nitrogen accumulation. The relationships of eight vegetation indicators and leaf nitrogen accumulation were analyzed using statistical models. Hyper-spectral variables were significantly correlated with leaf nitrogen accumulation, and the relationships of leaf nitrogen accumulation to SDr/SDb, FD742 and AVHRR-GVI were all highly significant with determination of coefficients (R2) as 0.9163, 0.9097 and 0.9142, respectively, and standard errors (SE) as 1.165, 1.079 and 1.077, respectively. Tests with another independent dataset showed that FD742 and REPIG could well predict leaf nitrogen accumulation in wheat with an R2 of 0.8449 and 0.8394, and root mean square error (RMSE) of 0.984 and 1.014, respectively. This suggests that FD742 and REPIG can be used to estimate leaf nitrogen accumulation, of which FD742 performed best in modeling and testing.