India has the highest burden of both tuberculosis (TB) and Multidrug Resistant tuberculosis (MDR-TB), based on estimates reported in Global TB Report 2016. Conventional diagnostic techniques are less sensitive & time consuming. The Xpert MTB/RIF Assay has the capacity to revolutionize the diagnosis of TB because of its speed, sensitivity and specificity. The objectives of our study are to determine the rate of positivity of Mycobacterial tuberculosis complex among the different clinical specimen and to know the rate of Rifampicin resistance among these specimens by using Xpert MTB/RIF Assay. The study was conducted in Department of Microbiology, Rural Medical College, (PIMS), Loni. Clinical Samples from suspected TB cases were treated according to Manufacturer’s Instructions & Standard operating procedure. A total of 1528 Samples received during the study period out of which 267 (17.47%) were positive for complex (MTBC) and 15 (0.98%) were rifampicin resistant. Maximum positivity was noted in age group 21-30 years. Out of total 126 extrapulmonary specimens, 10 were positive for MTBC. Also out of 273 HIV infected cases 16 samples were positive for MTBC while 1 was Rifampicin resistant.Accurate early diagnosis and treatment of TB has the potential to reduce morbidity and mortality associated with MDR TB. Xpert MTB /RIF assay has brought revolution in the earliest diagnosis (Turn around time-2.5hrs) compared to culture (which takes weeks) with simultaneous drug susceptibility testing.