The knowledge of root canal curvature is crucial regarding the prevention of ledge formation, root perforation and the possibility of endodontic instruments' fracture during endodontic treatments. Therefore, a quantification method of the root canal curvature as well as the applicability of diagnostically relevant tomographic three-dimensional (3D) imaging data is necessary. Hereby, cone-beam computed tomography (CBCT) images and micro-computed tomography (mu CT) data of distal root canals were analysed concerning the continuous three-dimensional curvature of human mandibular molars (n = 50). The curvature of the canal's three-dimensional centre line was determined by evaluating the tomographic images. The centroids of each root canal slice were identified and approximated by spline curves to obtain the centre line and therefore, its curvature. Comparing the results evaluated from CBCT and mu CT images, minimum radii of curvature of 2.6 mm and 2.1 mm were determined, respectively. The observation of the centre line demonstrated the requirement of the three-dimensional imaging data from CBCT and mu CT for a reliable curvature analysis. Conclusively, the evaluation of CBCT and mu CT images results in comparable radii of curvature. Thus, the application of the introduced method in combination with CBCT applied to patient cases could offer an important preliminary diagnostical step to prevent endodontic treatment complications.