We studied the rifampicin (RIF) and isoniazid (INH) resistant genes in Mycobacterium tuberculosis isolated from sputum samples of re-treated TB cases using phenotypic drug susceptibility test (DST) and rapid molecular method (Genotype; MTBDR plus, Hains Life Science, Nehren, Germany). Of the total 207 isolates included in this study, 90 (42.0%) were RIF resistant, 107 (50.7%) were INH resistant whereas 86 (41.5%) were multidrug resistant (MDR) by DST. According to genotypic analysis, 37.0% (77/207) were RIF resistant, 50.2% (105/207) INH resistant and 70 (33.8%) were MDR. Among the total 90 RIF resistant isolates, 85.5% (77/90) showed mutation in 81 bp of rpoB gene and the mutation locus were distributed as follows; MUT1 Asp516Val (16.7%; 15/90), MUT2A His526Tyr (6.6%; 6/90), MUT2B His526Asp (5.5%; 5/90) and MUT3 Ser531Lue (56.7%; 51/90). Among the total 107 INH resistant isolates, the mutation located in katG gene was 98.1% (105/107) and in inhA MUT1 gene was (16.8%; 18/107). The mutation locus were distributed as follows; MUT1 Ser315Thr (77.6%; 83/107), MUT2 Ser315Ile (3.7%; 4/107) in katG and in inhA MUT1Cys15Thr (16.8%; 18/107). The highest frequency of mutations in rpoB gene was found at the region of codon Ser531Leu (56.7%) while the frequency of mutation in katG and inhA gene were found at the region of codon Ser315Thr (77.6%) and Cyst15Thr (17.1%), respectively. The rapid molecular test kit used in this study was found to be sensitive as well as specific for detection of RIF and INH resistant gene and also helpful in early detection of MDR-TB.