Introduction: The study objective was to evaluate the effectiveness of cone-beam computed tomographic (CBCT) imaging for the detection of lateral canals (LCs) in endodontically treated premolars. Methods: Two evaluators classified 80 extracted premolars into 2 groups based on the absence (n = 40) or presence (n = 40) of LCs according to micro computed tomographic analysis. The extracted teeth were fixated in a human mandible and scanned with CBCT imaging. Subsequently, each tooth was endodontically treated, and CBCT scans were repeated. Three experienced examiners evaluated all images randomly. Receiver operating characteristic curves were compared using the McNemar test, and sensitivity, specificity, positive predictive value, and negative predictive value (NPV) were obtained. Results: The area under the receiver operating characteristic curve values were 0.58 and 0.49 before and after root canal treatment, respectively. These values were statistically significantly different (P < .001). Before root canal treatment sensitivity, specificity, positive predictive value, and negative predictive value were 55%, 52%, 55%, and 56%, whereas after root canal treatment the values were 33%, 61%, 46%, and 48%, respectively. Conclusions: LC detection in nontreated teeth presented low accuracy, whereas among treated teeth CBCT imaging showed no efficacy. The results suggest that CBCT imaging is not an effective diagnostic tool for LC detection.