To develop a stable estimation model and identify effective wavelengths that could explain the variations in leaf nitrogen (N) concentration with different N supplies, growing seasons, ecological locations, growth stages, and wheat cultivars. Four field experiments were performed during two consecutive years (2017–2019) at three sites (Yuanyang, Hebi, and Wenxian) in Henan, China. In situ canopy spectral reflectance data under the aforementioned N supply conditions were obtained over a range of 400–950 nm (visible and near-infrared region). On the basis of the canopy raw spectral reflectance data and their subsequent transformation by two different techniques, first-derivative reflectance (FDR) and continuum removal (CR), four multivariate regression methods were comparatively analyzed and used to develop predictive models for estimating leaf N concentration: multiple linear regression (MLR), principal component regression (PCR), partial least square (PLS), and support vector machine (SVM). Results showed that leaf N concentration and canopy reflectance significantly varied with the levels of N fertilization, and a good correlation was observed for all the spectral techniques. Seven wavelengths with relatively higher r values than the bands of the raw spectra centered at 508, 525, 572, 709, 780, 876, and 925 nm were specified using the FDR technique. Based on the full wavelengths, the FDR-SVM model exhibited a good performance for leaf N concentration estimation, with coefficients of determination ( r 2val) for the validation datasets and corresponding relative percent deviations (RPDval) values of 0.842 and 2.383, respectively. However, the FDR-PLS yielded a more accurate assessment of the leaf N concentration than did the other methods, with r 2val and RPDval values of 0.857 and 2.535, respectively. The variable importance in projection (VIP) scores from the FDR-PLS with the all canopy spectral region were used to screen the effective wavelengths of the spectral data. Therefore, six effective wavelengths centered at 525, 573, 710, 780, 875, and 924 nm were identified for leaf N concentration estimation. The SVM regression method with the effective wavelengths showed excellent performance for leaf N concentration estimation with r 2val = 0.823 and RPDval = 2.280. These results demonstrated that the in situ canopy spectral technique is promising for the estimation of leaf N concentration in winter wheat based on the FDR-PLS regression model and the effective wavelengths identified.