Untreated isthmuses can be a cause of endodontic treatment failure. We investigated the anatomic features of the isthmus in the mesial root of mandibular first molars using microcomputed tomography scans. Thirty-six extracted mandibular first molars were collected from the Chinese population and divided into three age groups as follows: 20 to 39 years (group A), 40 to 59 years (group B), and > or = 60 years (group C). Each tooth was scanned and reconstructed, and then the prevalence and type of isthmus were recorded. The percentage of sections showing isthmuses for groups A, B, and C were 50%, 41%, and 24%, respectively. The chi-square test indicated a significant correlation of the distribution of isthmuses with age (p < 0.001). The ratio of partial isthmus to complete isthmus for group C (17.1:1) was significantly higher than group A (5.9:1) and group B (7.0:1) (p < 0.001). By understanding the configuration and location of isthmus, a more efficient endodontic microsurgery can be guaranteed.