The purpose of this investigation was to evaluate the usefulness of multiplex polymerase chain reaction (m-PCR) in detecting uterine tuberculosis in women with infertility. In a prospective study, endometrial curetting from 620 females with infertility were investigated using laparoscopy, hysteroscopy, histopathology, smear microscopy, mycobacterial culture in BACTEC MGIT™ 960, and in-house m-PCR. The mean age of the women was 29.75 ± 4.66 years. The majority (596) sought medical attention for infertility; of them, 455 (76.34 %) presented with primary and 141 (23.65 %) with secondary infertility. A total of 158 (25.48 %) women were diagnosed as having uterine tuberculosis by at least one of the diagnostic methods. Among them, laparoscopy was positive in 46 (29.11 %), hysteroscopy in 77 (48.73 %), histopathology in only 8 (5.06 %), smear for acid fast bacilli in 4 (2.53 %), and liquid culture in 24 (15.18 %) patients. The in-house m-PCR was positive in 135 (85.44 %) women. Of these, 129 (95.55 %) samples were positive for Mycobacterium tuberculosis, while 6 (4.44 %) were positive for non-tuberculous mycobacterial DNA. Of the 129 M. tuberculosis PCR-positive women, 112 received anti-tubercular treatment and 23 of these conceived and fell pregnant after the completion of treatment. For the diagnosis of uterine tuberculosis, m-PCR was found to be the most efficient diagnostic tool compared to the other methods.