Background: Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), which remains the leading cause of morbidity and mortality worldwide. The emergence of drug-resistant strains of MTB has put status of TB to threatening levels. Aim of the Work: was to detect MTB along with rifampicin (RIF) resistance using Genexpert (MTB/RIF). Its diagnostic, sensitivity and specificity were evaluated by comparing with conventional technique. Patients, Materials and Methods: This prospective study was conducted on two hundred and seven Egyptian patients at Abbasia Chest Diseases Hospital, from November 2016 to December 2017, and comprised clinically and radiologically diagnosed TB suspected cases. This study was approved by the Ethical Committee of faculty of medicine, Alazhar University and the Ethical Committee of Ministry of Health and after Verbal consents from the patients or their parents were taken. Pulmonary specimens (sputum and bronchial lavage) and pleural effusion as an extra-pulmonary specimen were included. All samples collected were sent to TB laboratory of Abbasia Chest Diseases Hospital for further analysis. Result: Out of the 26 Genexpert (MTB/RIF) positive samples for MTBC, 3 (11.54%) showed RIF resistance and diagnosed as MDR-TB. Using LJ and MGIT cultures for drug sensitivity test (DST) on 31 and 34 positive TB samples; respectively, the same three specimens showed resistance to rifampicin (RIF). Four positive specimens were also resistant to streptomycin (STR) using the previously mentioned cultures. Moreover, resistant to INH was reported in five positive TB samples using the same cultures. Finally, it was found that all positive specimens were sensitive to Ethambutol (ETH). Conclusion: Although the conventional methods remain the gold standard for diagnosing pulmonary TB, delayed diagnostic times demand for more rapid and sensitive nucleic acid amplification techniques. Genexpert (MTB/RIF) assay is simple, rapid and accurate method for detecting mycobacterial tuberculosis