Aim: This study aimed to analyze incidence of three canals in human permanent maxillary first premolar teeth using cone beam tomographic techniques. Materials and Methods: The samples were divided into three groups namely Group (GP) I (single rooted [ n = 255]), GP II (two rooted [ n = 326]), and GP III (three rooted [ n = 8)]. The teeth were processed, mounted in arches, coded, and subjected to cone beam tomographic scanning. Incidence of three canals was evaluated by cone beam tomography, compared, and statistically analyzed. Results: The percentage of incidence of three canals in human permanent maxillary first premolar teeth was 1.7% and there was no statistically significant difference of incidence of three canals between studies using cone beam and non-cone beam methodologies. Conclusion: Clinicians should be aware of the variations they may encounter when endodontically treating a maxillary first permanent premolar and should apply this knowledge in a clinical scenario systematically.