Objective: The aim of this study was to evaluate the benefits of using two types of samples: the endometrial biopsy (EB) and the pouch of Douglas (POD) fluid by real-time polymerase chain reaction (PCR) for an early and accurate diagnosis of female genital tuberculosis (FGTB). Materials and methods: Two samples, one EB and second POD fluid, from each patient (total 173 patients) were subjected to IS6110-based real-time PCR assay for the diagnosis of FGTB. Results: Among 173 patient samples received, a total of 32.94% (57/173) patients were positive for MTB by either or both sample types. Endometrial biopsy samples were positive in 19.60% (34/173) of all EB samples for MTB, and POD fluid samples were positive in 24.27% (42/173) of all POD fluid samples for MTB. Among the total MTB-positive patients, 26.31% (15/57) patients had only EB sample as positive, whereas in 40.35% (23/57) patients only POD samples were positive. Conclusion: When genital tuberculosis is suspected clinically, it is better to opt for performing less invasive procedure like testing of POD fluid for MTB, and if it is negative, then further invasive procedure to obtain EB may be chosen for diagnosis.