Purpose: To analyze prevalence of mutations in genes associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates from patients with possible MDR TB of Puducherry, South India and to explore the association of specific mutations conferring rifampicin (RIF) resistance. Methods: We performed a commercial Genotype MDBDRplus V.2.0 assay for the rapid detection of rifampicin and isoniazid resistance directly on sputum specimens of patients with possible MDR TB. Results: Totally 558 multidrug resistant, 293 RIF mono resistant and 923 INH mono resistant tuberculosis were detected from the 12,786 patients with possible MDR TB samples. The 50.5% mutations were observed in the region of S531L in MDR TB patients and 55.6% in rifampicin monoresistant cases. In total isoniazid monoresistant, 68.0% mutations were detected in katG gene, which is more prevalent in comparison to inhA gene 32.0%. There were about 57.9% and 32.2% MDR TB cases diagnosed in the age group of > 15 to ≤ 45 years and > 45 to ≤ 60 years respectively. Conclusions: The rate of occurrences of mutations were found widely in the Rifampicin Resistant Determination Region (81 bp) of rpoB gene and the hypervariable region 530-533 codons of rpoB gene is alarming in the specification. The higher frequency of mutation in codons of rpoB (S531L) and katG (S315T) gene help to design simple, new and less expensive molecular techniques to use in peripheral laboratories.