Introduction: Multidrug-resistant tuberculosis (MDR-TB) is an emerging public health problem in many regions of the world, particularly in developing nations. Accurate and rapid diagnosis is essential in the management of MDR-TB, not only to optimize treatment but also to prevent transmission. Aims: To evaluate drug resistance in culture isolates by conventional and molecular methods and detect drug resistance gene in MDR-TB patients. Material and Method: 100 newly diagnosed pulmonary tuberculosis (TB) diagnosed patients attending TB Clinic, Gandhi Hospital, Secunderabad were included in the study. Two sputum samples collected from the patients were subjected to sputum microscopy, culture, Drug Susceptibility Testing (DST). Geno Type Mycobacterium Tuberculosis Drug Resistance (MTBDR) plus assay was done on the culture isolates to detect Rifampicin and Isoniazid (INH) resistance. Results: Out of 100 samples, 48 % smear positivity by Ziehl Neelsen (ZN) method, 51 % culture positivity on LJ medium,11.7% multi drug resistance for Rifampicin and Isoniazid with conventional drug susceptibility – Proportion method,17.6 % drug resistance by molecular method – Geno Type MTBDR plus was observed. Among the 4 Rifampicin (Rif) resistant isolates 2isolates showed mutation (mut) at D516V and in other 2 isolates only wild type (WT) was missing but no mut was seen . In the 1 Isoniazid (INH) resistant isolate WT was missing, but no mutation was seen. Among the 4 Rif +INH resistance all showed mut at S531L for RIF and at S315T1. Conclusion: The Genotype MTBDR assay is a rapid and reliable tool for the routine direct detection of MTB strains and of strains resistant to INH and RIF in smear positive, highly infectious patients. The rapid turn around time of the test enables the optimization of the therapy of these patients before confirmatory culture results are available. The test does not require viable organisms and thus reduces the biohazard risk in the laboratory.