We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for the early detection and identification of Mycobacterium tuberculosis directly from clinical samples. PCR-RFLP of hsp65 was applied to the DNA extracted directly from sputum samples (n = 226) collected from 226 patients. We could detect and identify M. tuberculosis in 84.5% of the acid-fast bacillus (AFB) smear-positive samples (n = 149) and 11% of the AFB smear-negative samples (n = 18) obtained from patients with clinical and radiological evidence of tuberculosis. Sputum samples (n = 59) obtained from patients suffering from respiratory diseases other than tuberculosis were included as negative controls. To test the sensitivity of the assay, we spiked a smear-negative sample with serial dilutions of H37Rv. The protocol could detect down to 10 organisms/microl. PCR-RFLP was found to be a simple and reproducible method for early detection of M. tuberculosis from sputum samples. The present assay could be used to augment conventional methods of diagnosis of mycobacterial diseases and thus help clinicians to differentiate between M. tuberculosis complex and non-tuberculous mycobacteria directly in clinical samples. The assay could help clinicians to select appropriate chemotherapeutic agents early, which could considerably reduce the morbidity due to mycobacterial diseases.