BACKGROUND AND AIM: Isthmuses are narrow and strip shaped connections between two canals containing pulpal tissue that may encompass bacteria as well. They are hardly accessible for cleaning; therefore, success and failure of root canal treatments could be affected by their presence. The present study was carried out aiming to evaluate the incidence, location, and type of isthmus in mandibular second molars (MSMs) among individuals of an Iranian population.METHODS: 80 MSMs with inclusion criteria, belonging to individuals of an Iranian population were collected and disinfected. After embedding the samples’ roots in the acrylic resin, teeth were sectioned horizontally at 2, 4, and 6 mm distances from the apices. Then they were viewed and recorded by a stereomicroscope under × 30 magnification. According to Kim classification, two endodontists recorded the number of canals, incidence, and type of the isthmuses. In cases of disagreement, a third endodontist was involved to judge on the right opinion between the two. The acquired data were analysed using chi-square test.RESULTS: The results showed that 96.3% and 3.75% of MSMs had two and three roots, respectively. Type V (described in the following) was the most frequent type of isthmus at mesial and distal roots. C-shaped canals composed 3.7% of the samples and showed isthmus at all the three sections. There was a significant difference in the distribution of isthmuses (P < 0.001).CONCLUSION: According to this study, different types of isthmuses prevail in high rate of mesial and about half of the distal roots of MSMs, which are important for root canal cleaning and shaping.