Histopathological and mycobacteriological examinations have limited utility in the diagnosis of genital tuberculosis. In this double-blind study, 61 samples, consisting of endometrial aspirates (EAs), endometrial biopsies (EBs) and fluid from the pouch of Douglas (POD), from 25 women suffering from infertility were investigated for the presence of the mpt64 gene of Mycobacterium tuberculosis by PCR and correlated with laparoscopic findings. PCR demonstrated M. tuberculosis DNA in 14 out of 25 patients (56.0 %), compared to one smear with acid-fast bacilli (1.6 %) and two culture-positive samples (3.2 %). The presence of M. tuberculosis DNA was observed in 53.3 % of EBs, 47.6 % of EAs and 16.0 % of POD fluid samples. All patients with laparoscopy suggestive of tuberculosis, 60 % of those with a probable diagnosis and 33 % of those with incidental findings were positive by PCR. However, one EA sample from an infertile patient with normal laparoscopy was also positive. Multiple sampling from different sites and amplification of the mpt64 gene segment by PCR offered increased sensitivity in determining tuberculous aetiology in female infertility.