AbstractAlthough teeth microcracks (MCs) have long been considered more of an aesthetic problem, their exact role in the structure of a tooth and impact on its functionality is still unknown. The aim of this study was to reveal the possibilities of an X-ray micro-computed tomography ($μ$ μ CT) in combination with convolutional neural network (CNN) assisted voxel classification and volume segmentation for three-dimensional (3D) qualitative analysis of tooth microstructure and verify this approach with four extracted human premolars. Samples were scanned using a $μ$ μ CT instrument (Xradia 520 Versa; ZEISS) and segmented with CNN to identify enamel, dentin, and cracks. A new CNN image segmentation model was trained based on “Multiclass semantic segmentation using DeepLabV3+” example and was implemented with “TensorFlow”. The technique which was used allowed 3D characterization of all MCs of a tooth, regardless of the volume of the tooth in which they begin and extend, and the evaluation of the arrangement of cracks and their structural features. The proposed method revealed an intricate star-shaped network of MCs covering most of the inner tooth, and the main crack planes in all samples were arranged radially in two almost perpendicular directions, suggesting that the cracks could be considered as a planar structure.