Featured Application: The digital analysis of hyperspectral images by means of artificial intelligence can contribute to the development of new diagnostic techniques for early caries detection. Stains and stained incipient lesions can be challenging to differentiate with established clinical tools. New diagnostic techniques are required for improved distinction to enable early noninvasive treatment. This in vitro study evaluates the performance of artificial intelligence (AI)-based classification of hyperspectral imaging data for early occlusal lesion detection and differentiation from stains. Sixty-five extracted permanent human maxillary and mandibular bicuspids and molars (International Caries Detection and Assessment System [ICDAS] II 0–4) were imaged with a hyperspectral camera (Diaspective Vision TIVITA^® Tissue, Diaspective Vision, Pepelow, Germany) at a distance of 350 mm, acquiring spatial and spectral information in the wavelength range 505–1000 nm; 650 fissural spectra were used to train classification algorithms (models) for automated distinction between stained but sound enamel and stained lesions. Stratified 10-fold cross-validation was used. The model with the highest classification performance, a fine k-nearest neighbor classification algorithm, was used to classify five additional tooth fissural areas. Polarization microscopy of ground sections served as reference. Compared to stained lesions, stained intact enamel showed higher reflectance in the wavelength range 525–710 nm but lower reflectance in the wavelength range 710–1000 nm. A fine k-nearest neighbor classification algorithm achieved the highest performance with a Matthews correlation coefficient (MCC) of 0.75, a sensitivity of 0.95 and a specificity of 0.80 when distinguishing between intact stained and stained lesion spectra. The superposition of color-coded classification results on further tooth occlusal projections enabled qualitative assessment of the entire fissure’s enamel health. AI-based evaluation of hyperspectral images is highly promising as a complementary method to visual and radiographic examination for early occlusal lesion detection.