Introduction Fistulae-in-ano with a specific aetiology such as TB and Crohn's are usually complex and challenging to treat. This study was aimed to determine the yield of routine histological analysis in fistula-in-ano, in detecting specific aetiology. Methods A descriptive study was conducted at the Professorial Surgical Unit, National Hospital of Sri Lanka, Colombo from 2001-2016. Histopathology reports of all patients without a histological diagnosis and who underwent surgery for fistulae-in-ano were analysed. Results A total of 215 patients [median age:40 years (range:14-73), males=179(82.8%)] were analysed. The majority (75%, n=161) were simple fistulae. Histological evaluation revealed inflamed granulation tissue in 94.9% (n=204) of patients. Five (2.3%) patients had conclusive evidence of Crohn's disease and three (1.4%) had tuberculosis. One patient (0.5%) had evidence of adenocarcinoma with mucinous differentiation. Significant proportion of fistula with underlying specific aetiology were complex fistulae (82% vs. 22%, p<0.001) and associated with abscess/collections (45.5% vs. 11.8%, p<0.001). Age, type of fistula, level of internal opening, recurrence and presence of haemorrhoids were comparable in those with and without a specific aetiology. Conclusions Complex fistula and the presence of abscess/ collection were associated with a specific aetiology. Routine histopathological analysis in patients presenting with fistula in-ano should be performed as a clinical prediction based on the nature of fistula may not be always accurate.