Purpose: To evaluate diagnostic accuracy of IS6110 insertion genes, hsp65, and Xpert MTB/RIF for rapid diagnosis of pulmonary tuberculosis. Methods: Sixty patients, medically reported HIV negative, clinically suspected of having pulmonary tuberculosis, were included in this study, and consented before enrolment. Sputum samples were gathered once, and tested by smear for Acid Fast Bacilli (AFB). Cultured in the Loewenstein-Jensen (LJ) medium for M. tuberculosis growth, M. tuberculosis DNA was detected by conventional PCR targeting IS6110, and hsp65 genes using specific primers, and automated nested real-time PCR targeting rpoB gene. Sensitivity, specificity and diagnostic accuracy were calculated for each method compared to culture. Results:Compared with culture as reference method, smear, IS6110, hsp65, and Xpert MTB/RIF had sensitivity 77.14%, 100%, 100%, and 100%, specificity 92%, 96%, 96%, and 96.97%, and diagnostic accuracy 83.33%, 98.33%, 98.33% and 98.21% respectively. Molecular diagnostic methods had the highest diagnostic accuracy, whereas smear had the lowest. No statistical significance, (p value > 0.05) was detected between the patients' demographic data and the presence or absence of TB infection. Conclusion: The diagnostic accuracy that we got from the molecular methods, confirmed the diagnostic value of molecular detection of M. tuberculosis in pulmonary cases, supporting the application of automated and conventional PCR in rapid analysis. Smear could be more efficient when used for treatment monitoring. Combination between one-molecular techniques with smear as a routine method could be valid for rapid diagnosis of TB.