Purpose To retrospectively assess the accuracy of magnetic resonance imaging (MRI) in defining dentate line in anal fistula. Materials and methods Seventy patients with anal fistulas were assessed by dynamic contrast-enhanced MRI. The distance from the dentate line to the anal verge for all patients was measured by MRI. To mitigate interference, 35 patients with anal fistulas whose internal openings were located on the dentate line were excluded from this study. Two observers independently judged the positional relationship between the internal opening and the MRI-defined dentate line, and compared with the results observed by surgeon to assess the accuracy. Results The distance between the MRI-defined dentate line and the anal verge depended on the location of the internal opening and the morphology of the anal canal mucosa. The distance based on the location the internal opening and the morphology of the anal canal mucosa was 18.2 +/- 8.1 mm and 20.0 +/- 5.3 mm on oblique coronal T2WI, respectively. Compared with the results observed by the surgeon, the accuracy of evaluating the positional relationship between the internal opening and the dentate line from the morphology of the anal canal mucosa on MRI exceeded 89.9%. Taking 18.2-20.0 mm as the distance between the dentate line and the anal verge on the MRI image, the accuracy of evaluating the relationship between the position of the internal opening and the dentate line was over 85.7%. Considering both the dentate line and the anal canal mucosa, the accuracy of evaluating the relationship between the internal opening and the dentate line was over 91.5%. The results of MRI-defined dentate line were in good agreement with the results of intraoperative surgeon evaluation, and the kappa values were 0.70, 0.63, and 0.78, respectively. Conclusion MRI has high accuracy in defining the dentate line in anal fistulas.