Introduction: The isthmus is a communication between two or more root canals that contains pulp tissue. Its unpredictable location, variable prevalence, and anatomic complexity often result in challenging debridement. The knowledge of these factors may prove beneficial for successful endodontic treatment. The primary aim of this systematic review was to assess the prevalence and morphology of root canal isthmus (RCI) in human permanent teeth using micro-computed tomography (micro-CT) studies. The secondary aim was to assess the influence of demographic factors such as age, gender, and geographic region on the prevalence and morphology of the isthmus. Materials and Methods: An electronic search was conducted until January 2020. Studies published in the English language using micro-CT for the evaluation of isthmus prevalence and morphology were included. Sixteen articles met the inclusion criteria and were subjected to qualitative synthesis. Results: The prevalence of isthmus in the mesiobuccal root of maxillary first molars ranged from 8.9% to 70.8%, with maximum prevalence in the middle third of the root (49.50%–73%). The mesial root of mandibular molars had a prevalence ranging from 80.6% to 100% on full root analysis and 85%–88.89% in the apical third. Conclusion: The prevalence of RCI was higher in the mesiobuccal roots of maxillary molars and the mesial roots of mandibular molars, extending in the corono-apical direction with a complex and variable morphology. The influence of demographic factors on the prevalence of RCI could not be established clearly.