Background: The diagnosis of nondisplaced longitudinal fractures [vertical root fractures (VRFs)] is challenging in clinical practice. Radiographic techniques showed a difficulty in detection of VRFs. Cone beam computed tomography (CBCT) is a new diagnostic imaging modality that provides high-quality three-dimensional (3D) images for dental diagnosis. Aims: The aim of this in vitro study is to compare accuracy of three different imaging modalities: conventional periapical radiographs, digital radiographs, and CBCT in detecting VRFs in teeth that are endodontically as well as non-endodontically treated. Materials and methods: An in vitro model consisting of 60 recently extracted human mandibular lower premolars were used. Root canal treatment was carried out for 30 teeth. Root fractures were created in 30 teeth (15 root canal treated and 15 non-treated) by mechanical force. Other 30 teeth remain intact. The teeth were mounted and images were taken with a periapical, digital, and CBCT X-ray unit. Three endodontists separately evaluated the images. Results: Interobserver κ values showed a very good interobserver agreement (0.98 for CBCT, 0.88 for digital, and 0.93 for conventional periapical X-rays). There was an overall statistically significant difference (p = 0.00) in detecting of root fracture among the three imaging modalities and the highest accuracy with CBCT images. Conclusions: In in vitro model, CBCT scan appears to give the highest accuracy in detecting VRFs when compared with the periapical systems in both endodontically and non-endodontically treated teeth. Clinical significance: The CBCT scan shows higher sensitivity in detection of VRFs in comparison with periapical images.