Objectives: This study evaluated the accuracy of cone-beam computed tomography (CBCT) in detecting the root canal morphology of mandibular first premolars using micro-computed tomography (micro-CT) as a reference standard. Materials and methods: In total, 143 extracted human mandibular first premolars were selected and scanned using micro-CT and CBCT. The acquired images were used to evaluate the root canal morphology in each tooth, and evaluations were repeated after 2 weeks. The root canal configurations observed on the three-dimensional images were recorded, and the findings from both modalities were compared using chi-square tests. The actual agreement between the two modalities was assessed using kappa statistics. Results: In total, the root morphologies in 136 mandibular first premolars were consistently identified by both CBCT and micro-CT: type I in 104, type III in five, type V in 20, and type IX in seven. Of the remaining seven teeth, the morphology in two, one, and four teeth was identified as type I, type VII, and type IX (type 1-3 in two and type 1-2-3 in two), respectively, by micro-CT and misdiagnosed as type III, type V, and type V, respectively, by CBCT. There were no significant differences between the two modalities with regard to the accurate detection of root canal configurations, with a kappa value of 0.886 for the actual agreement. Conclusions: Although CBCT may be accurate in detecting the root canal configuration in mandibular first premolars, it produces poorer image details compared with micro-CT. Clinical relevance: CBCT is a reliable radiological technique, but its accuracy in detecting details of the root canal morphology in mandibular first premolars, especially in some complex root canal configurations, needs to be improved. [ABSTRACT FROM AUTHOR]