This study aimed to determine the role of magnetic resonance imaging (MRI) in diagnosing and describing the characteristics of fistula-in-ano, and the agreement between MRI and operative findings. We conducted a retrospective study in 367 patients with fistula-in-ano who were diagnosed and had an operation at the University Medical Center between January 2016 and January 2018. MRI findings were evaluated and compared with surgical findings using the kappa coefficient (k) method. 367 patients (327 male and 40 female, mean age 39.3 ± 12.4 years). A total of 411 primary fistulas were found during surgery. There was a strong agreement between MRI and surgery for classifying primary tracts (k = 0.89) and detecting secondary tracts (k = 0.94). While the sensitivity and specificity of MRI for detecting internal openings were 99% and 85.2% respectively; these rates were 100% for abscesses. Both T2-weighted turbo spin-echo (T2W TSE) and postcontrast fat-saturated T1-weighted turbo spin-echo (FS T1W TSE) sequences showed high sensitivity (96.6% and 98.4% respectively) and specificity (92.6% and 81.5% respectively) for depicting internal openings and secondary tracts. Post-contrast FS T1W TSE sequence was very effective in detecting abscesses with an accuracy of 100%. In conclusion, MRI can be considered an accurate tool for the preoperative evaluation of fistula-in-ano, which is a major determinant of the surgical outcome. Both T2W TSE and post-contrast FS T1W TSE sequences are highly accurate in depicting the features of fistula-in-ano. If there are no contraindications, contrast administration is recommended to differentiate abscesses from active inflammation.