Background: This study aimed to assess the diagnostic accuracy of cone beam computed tomography (CBCT) and quantitatively evaluate the morphology of mandibular first molars using CBCT. Material and Methods: Twenty-four double-rooted mandibular first molars were evaluated by NewTom VGi CBCT. The distance from the furcation and apex to the cementoenamel junction (CEJ), diameter and thickness of canal walls, the buccolingual (BL) to mesiodistal (MD) ratio (ΔD), prevalence of oval canals at different sections and taper of the canals were all determined. In order to assess the diagnostic accuracy of CBCT, distance from the furcation and apex to the CEJ and thickness of canal walls at the CEJ and apex were compared with the gold standard values (caliper and stereomicroscope). Statistical analyses were carried out using intraclass correlation coefficient (ICC), paired t-test and repeated measures ANOVA. Results: A high correlation existed between the CBCT and gold standard measurements ( P <0.001). In dimensional measurements, length of mesial root was higher than the distal root and lingual furcation was farther from the CEJ than the buccal furcation ( P <0.001). An important finding of this study was the mesiodistal taper of the mesiobuccal (MB) and mesiolingual (ML) canals; which was equal to 0.02. Conclusions: CBCT has acceptable diagnostic accuracy for measurement of canal wall thickness. Cleaning and shaping of the canals should be performed based on the unique anatomy of the respective canal; which necessitates the use of advanced imaging techniques for thorough assessment of root canal anatomy in a clinical setting