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Background: Rapid and specific diagnosis of gastrointestinal tuberculosis (GITB) is of utmost importance. Aim: To evaluate Multiplex PCR (MPCR) using MPB64 and IS6110 primers specific for M. tuberculosis for rapid diagnosis of GITB. Materials and Methods: MPCR was performed on colonoscopy biopsy specimens on 11 GITB confirmed (culture/AFB/histopathology was positive), 29 GITB suspected and 30 Non GITB (control group) patients. Results: MPB64 PCR had sensitivity and specificity of 90% and 100% for confirmed GITB cases. In 29 clinically diagnosed but unconfirmed GITB cases, MPCR was positive in 72.41%. MPCR was negative in all control group patients. The overall sensitivity and specificity of microscopy, culture, histopathology and MPCR was 5%, 2% 20% and 77.5% and 100%, 100%, 100% and 100% respectively. Conclusion: MPCR has good sensitivity and specificity in diagnosing gastrointestinal tuberculosis.