Objective: The aim of the study was to evaluate the prevalence of root canal isthmus (RCI) and measure the interorifice distance (IOD) between the root canals. Additionally to correlate IOD with the RCI in human permanent teeth using conebeam computed tomography (CBCT) in an Indian sub-population (Chennai). Materials and Methods: A total of 5881 teeth from 280 CBCT full mouth scans were analyzed. The presence or absence of complete and incomplete RCI of each tooth was identified using the map-reading strategy. IOD was calculated by measuring the distance between the center of each root canal orifice to that of the center of the adjacent orifice at the level of the cementoenamel junction using the axial and sagittal sections. Chi-square analysis and correlation statistics using Spearman's rank-order test was done ( P < 0.05). Results: High prevalence of RCI was found in maxillary first premolars, mesial root of mandibular molars, and mesiobuccal root of maxillary first molars, while its prevalence was low in maxillary canines and mandibular premolars and absent in maxillary incisors. RCI was predominantly seen in the cervical and middle third of the root canal in all the teeth evaluated. A weak negative correlation was established between the IOD and RCI for maxillary premolars, whereas a weak positive correlation was seen in maxillary first molars and mandibular second molars. Conclusion: The prevalence of RCI was high in the posterior teeth in comparison to the anterior teeth in the present study. There was no strong correlation between IOD and RCI in all the teeth evaluated.