To evaluate the effect of low-temperature stress in winter wheat during the early growth stages, the response regularity of the canopy spectral reflectance was evaluated. Besides, winter wheat yield during the maturation stage and the relationship between yield and canopy spectral reflectance were also analyzed. Two multivariate methods, namely, the successive projections algorithm (SPA) and multiple linear regression (MLR), were combined to explore the relationship between the spectral reflectance and yield. Our results showed that the green peak and red valley in visible wavelengths altered obviously and the red edge gradually moved towards blue wavebands. The canopy spectral reflectance in the near-infrared wavebands increased with an increase in low-temperature stress intensity. Moreover, the reflectance proved that the red edge region under low-temperature stress is related to winter wheat yield, and approximately 38% of extracted wavebands were concentrated in the red edge region (680-780 nm). Compared with the predictive MLR models, the model calibrated during the flowering period of winter wheat (25 days post low-temperature treatment) had better performance in predicting crop yield. Whole-spectrum predictive models based on the principle component regression (PCR) method and Normalized Difference Vegetation Index (NDVI) models based on MLR were also established. Moreover, the performance of three kinds of calibration methods and the validation result of the field test were compared to select the optimal monitoring stage and technique to estimate the yield in the early growth stage of winter wheat under low-temperature stress. This study could provide a theoretical basis and practical reference for hyperspectral assessment of yield in winter wheat during low-temperature stress.