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Objective: The aim of this study was to determine the accuracy of three-dimensional endoanal ultrasound scan (3D-EAUS) in the pre-operative assessment of fistula-in-ano in identifying the fistula tract and comparing with findings at surgery in a South Asian cohort. A retrospective analysis of 87 patients with suspected fistula-in-ano who underwent pre-operative 3D-EAUS between January 2009 and January 2016 was carried out. All patients subsequently had surgical exploration under anaesthesia (EUA), irrespective of 3D-EAUS findings. The 3D-EAUS results were compared with the surgical findings to determine the accuracy of 3D-EAUS., Results: A total of 86 (98.9%) patients (male = 75) were subsequently shown to have a fistula at surgical exploration and of them, 3D-EAUS detected a fistula in 79 (92%) patients. In this cohort, 3D-EAUS correctly predicted the surgical findings in (n = 61, 70.9%) patients with the highest accuracy being for transphincteric fistulae (87.1%). However, the overall concordance in our study was low with a kappa coefficient of 0.318. Additional findings such as sphincter defects were detected by the 3D-EAUS in 37 patients (internal sphincter defects-21, external sphincter defects-7, both-9) which were not evident at EUA. Therefore, 3D-EAUS had a good accuracy in selected types of fistulae and particularly useful in identifying sphincter defects before surgery.