AbstractIn the agricultural industry, advances in optical imaging technologies based on rapid and non-destructive approaches have contributed to increase food production for the growing population. The present study employed autofluorescence-spectral imaging and machine learning algorithms to develop distinct models for classification of soybean seeds differing in physiological quality after artificial aging. Autofluorescence signals from the 365/400 nm excitation-emission combination (that exhibited a perfect correlation with the total phenols in the embryo) were efficiently able to segregate treatments. Furthermore, it was also possible to demonstrate a strong correlation between autofluorescence-spectral data and several quality indicators, such as early germination and seed tolerance to stressful conditions. The machine learning models developed based on artificial neural network, support vector machine or linear discriminant analysis showed high performance (0.99 accuracy) for classifying seeds with different quality levels. Taken together, our study shows that the physiological potential of soybean seeds is reduced accompanied by changes in the concentration and, probably in the structure of autofluorescent compounds. In addition, altering the autofluorescent properties in seeds impact the photosynthesis apparatus in seedlings. From the practical point of view, autofluorescence-based imaging can be used to check modifications in the optical properties of soybean seed tissues and to consistently discriminate high-and low-vigor seeds.