The development of non-destructive methods for early detection of cold stress of plants and the identification of cold-tolerant cultivars is highly needed in crop breeding programs. Current methods are either destructive, time-consuming or imprecise. In this study, soybean leaves’ spectra were acquired in the near infrared (NIR) range (588–1025 nm) from five cultivars genetically engineered to have different levels of cold stress tolerance. The spectra were acquired at the optimal growing temperature 27 °C and when the temperature was decreased to 22 °C. In this paper, we report the results of the aquaphotomics analysis performed with the objective of understanding the role of the water molecular system in the early cold stress response of all cultivars. The raw spectra and the results of Principal Component Analysis, Soft Independent Modeling of Class Analogies and aquagrams showed consistent evidence of huge differences in the NIR spectral profiles of all cultivars under normal and mild cold stress conditions. The SIMCA discrimination between the plants before and after stress was achieved with 100% accuracy. The interpretation of spectral patterns before and after cold stress revealed major changes in the water molecular structure of the soybean leaves, altered carbohydrate and oxidative metabolism. Specific water molecular structures in the leaves of soybean cultivars were found to be highly sensitive to the temperature, showing their crucial role in the cold stress response. The results also indicated the existence of differences in the cold stress response of different cultivars, which will be a topic of further research.