As an emerging cash crop, industrial hemp (Cannabis sativa L.) grown for cannabidiol (CBD) has spurred a surge of interest in the United States. Cultivar selection and harvest timing are important to produce CBD hemp profitably and avoid economic loss resulting from the tetrahydrocannabinol (THC) concentration in the crop exceeding regulatory limits. Hence there is a need for differentiating CBD hemp cultivars and growth stages to aid in cultivar and genotype selection and optimization of harvest timing. Current methods that rely on visual assessment of plant phenotypes and chemical procedures are limited because of its subjective and destructive nature. In this study, hyperspectral imaging was proposed as a novel, objective, and non-destructive method for differentiating hemp cultivars, growth stages as well as plant organs (leaves and flowers). Five cultivars of CBD hemp were grown greenhouse conditions and leaves and flowers were sampled at five growth stages 2–10 weeks in 2-week intervals after flower initiation and scanned by a benchtop hyperspectral imaging system in the spectral range of 400–1000 nm. The acquired images were subjected to image processing procedures to extract the spectra of hemp samples. The spectral profiles and scatter plots of principal component analysis of the spectral data revealed a certain degree of separation between hemp cultivars, growth stages, and plant organs. Machine learning based on regularized linear discriminant analysis achieved the accuracy of up to 99.6% in differentiating the five hemp cultivars. Plant organ and growth stage need to be factored into model development for hemp cultivar classification. The classification models achieved 100% accuracy in differentiating the five growth stages and two plant organs. This study demonstrates the effectiveness of hyperspectral imaging for differentiating cultivars, growth stages and plant organs of CBD hemp, which is a potentially useful tool for growers and breeders of CBD hemp.