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Introduction and Objectives Missed canal systems are the most frequent cause for endodontic treatment failure. The latter surely applies for mandibular molar teeth. In order to minimize the needs for retreatment, all potential diagnostic means should be applied in order to maximize visualisation and localisation of the root canal anatomy. The objective of the present study is to evaluate mandibular molar canal anatomy, with a specific focus on identification of a mesial root isthmus using microCT. Once this problem is fully understood, diagnostic CBCT imaging may be focused on identifying such structures. Method In this study 29 lower molar teeth were scanned using the SkyScan 1173 high-energy micro-CT system. The scan was made with an isotropic pixel resolution of 12.5 micron. For each sample, a stack of images was reconstructed providing axial cross sections of the internal structure of the samples. For every sample, a classification was made for the type of connection according to Hsu and Kim (1997). Results 3D surface rendered models show the complexity of the root canal system. The canals can end up separately (n= 12) or be fused ending in one single foramen (n=17). From the 29 samples, the following classification was made: 1 tooth type I, 2 teeth type II, 4 type III, 6 type IV and 16 teeth type V. Conclusion The study showed an important complexity and variability of canal morphology amongst mandibular molar teeth. With regard to the isthmus in the mesial root, isthmus type V was found in most of samples, suggestion full connection between 2 canals. The present findings on mandibular root canal morphology may further serve as gold standard to evaluate diagnostic image quality of CBCT images with a focus on canal and isthmus morphologic detection.