Introduction: The aim of the present study was to measure the percentage of the volume of the isthmus area of mesiobuccal root canal system of lower molars filled by a hybrid GuttaCore back filling technique using a high-resolution micro-computed tomography (µ-CT) scanning and to measure the amount of debris and root filling material in the isthmus after instrumentation/irrigation and root filling. Methods: Mandibular molars with two separated mesial root canals (16 teeth) were scanned by using the Skyscan 1172 m-CT system (Skyscan, Aartselaar, Belgium) before and after instrumentation and after filling using the Hybrid GuttaCore root filling technique. An isthmus was defined as the ribbon-shaped or thin connecting structures between two root canals after instrumentation. The characteristics of the isthmuses were quantitatively monitored during the whole treatment. The images were segmented and quantified. The surface area of the isthmus, volume of debris after rotary instrumentation, and volume of the filled space in the isthmus after obturation were evaluated. Results: Of the 16 mesial roots of the lower molars one tooth were excluded because of technical errors in scanning (because the samples mounted on the stubs were slightly moved during scanning process). 100% of all the isthmuses of the mesial canals of the lower molars in our project were filled either totally or partially. The small details of the canal and the anastomoses are beautifully filled out in several of the roots. None of the roots showed a total lack of filling material in the isthmus, but there were differences in the degree of obturation in between the canals. Conclusions: A considerable amount of dentin debris is produced and packed into the isthmus area during rotary instrumentation of mesial canals of lower molars despite continuous irrigation during and after instrumentation. The debris may partly prevent penetration of the filling material and sealer into the isthmus area.