Purpose: This study aimed to investigate inter-observer reliability among observers with different levels of proficiency and the diagnostic imaging reliability of cone-beam computed tomography (CBCT) images of the retromolar canal. Materials and Methods: CBCT images of 307 patients were assessed for the presence of retromolar canals (RMCs) by 3 observers independently. Diagnoses were made twice by each observer at intervals of more than 3 weeks. Inter-observer reliability was assessed using the kappa coefficient. One observer had no experience in diagnosis using CBCT images. Therefore, a specialist in diagnostic imaging explained the CBCT images for interpretation and practiced diagnostic imaging together with this observer, while the other observer interpreted the images independently. Thereafter, the observers re-evaluated the images. Results: The interobserver kappa coefficients (including bilateral RMCs) calculated at the first reading were low, ranging from 0.21 to 0.61. Their values ranged from 0.95 (right side) to 1.00 (left side) after one-on-one practice with a diagnostic imaging specialist, while the values ranged from 0.65 (right side) to 0.66 (left side) without one-on-one practice. Conclusion Diagnostic accuracy was improved through diagnostic imaging practice. To improve the anatomical interpretation of images, it is important to practice diagnostic imaging with a specialist in diagnostic imaging. One-on-one instruction about diagnostic imaging was an effective method of training.