India is high tuberculosis burdened country with increasing prevalence of multidrug resistant tuberculosis. Rapid diagnosis and prompt treatment of infectious cases are the key elements in reducing the spread of tuberculosis. In the present study we sought to ascertain multidrug resistant tuberculosis among previously treated tuberculosis cases and its resistance pattern to rifampicin and isoniazid genes. The samples from the patients of previously treated tuberculosis cases were collected from eleven districts of North Karnataka during July 2013 to December 2013 and transported to laboratory. The line probe assay was carried out on 265 smear positive samples to detect common mutations in the rpoB gene for rifampicin and katG and inhA genes for isoniazid, respectively. A total of 380 sputum samples from MDR suspects were received of which, 282 () isolates were found to be AFB Smear positive. All smear positive sample processed showed 102 () resistant to rifampacin and 107 ( %) resistant to Isoniazid. Missing wild type 8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates and missing wild type along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates. The MDR-TB among previously treated TB suspects tested in Northern Districts of Karnataka, India was found to be . The common mutations obtained for RIF and INH in the region was mostly similar to those reported earlier in different parts of India. | Molecular detection of Rifampicin and Isoniazid resistance and characterization of mutations in Mycobacterium Tuberculosis complex using line probe assay