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The purpose of this study was to assess the accuracy of 2-dimensional morphometric parameters of root canals on different cone-beam computed tomographic (CBCT) images using 2 segmentation methods (operator dependent and Otsu's automatic), considering micro–computed tomographic (micro-CT) images as the reference standard.Ten mandibular molars were scanned by micro-CT imaging and 3 different CBCT devices: Accuitomo (J Morita Corporation, Kyoto, Japan), NewTom 5G (CEFLA, Imola, Italy), and NewTom VGi evo (CEFLA). The images were standardized and recorded using MeVisLab software (MeVis Medical Solutions AG, Bremen, Germany). Two calibrated examiners assessed the images of axial reconstructions quantitatively by 2-dimensional parameters (area, perimeter, roundness, and largest and smallest diameter). Fleiss kappa was performed to check interrater and intrarater reliability. The absolute error was calculated as the means and standard deviation. One-way analysis of variance was performed for comparison between the methods used by the operator and Otsu's automatic thresholding. To determine the accuracy of CBCT devices, the absolute error rate of each parameter was calculated using micro-CT measurements as the reference value with thresholding determined by the operator.The thresholding method performed by the operator had lower absolute error values for area, perimeter, and major and minor diameters, differing (P < .05) from Otsu's automatic method, with no differences between the CBCT machines.An overestimation of area, roundness, and major and minor diameters and an underestimation of the perimeter were shown for the 3 CBCT machines evaluated. Thresholding determined by the operator is more accurate than that determined by Otsu's automatic method for the assessment of 2-dimensional morphometric parameters, which could direct influence in the diagnosis and endodontic treatment plan.