Context: Rapid identification of resistant strains is crucial for the early administration of appropriate therapy and to curtail their spread. Genotype MTBDRplus is an assay for rapid detection of gene mutations in sputum samples. Aims: i) To detect the presence of rifampicin and isoniazid associated mutations directly in smear positive sputum samples. ii) Analysis of mutations in the region of North Coastal Andhra Pradesh. Materials and Methods: A total of 151 sputum samples were processed by N-acetyl L-cysteine - sodium hydroxide method. The sediment obtained was used for smear preparation, culture and nucleic acid extraction. Multiplex amplification and reverse hybridization were then performed according to manufacturer's instructions. The presence of a mutation is indicated by the lack of hybridization on one or more of wild-type probes with or without hybridization on the mutation probes. Results: Hundred percent readable results were achieved from sputum samples smear grade of 2+ and 3+. All strips had bands indicating Mycobacterium tuberculosis complex and were also confirmed by culture and biochemical tests. The strains resistant to rifampicin only, isoniazid only and multidrug resistant were 55(36.7%), 42(28%) and 40(26.7%) respectively. The most frequent type of mutation for rifampicin resistance was Serine-531-Leucine. The rate of mutations detected by negative hybridization results with wild-type probes was 17.5%. Conclusions: Potential for testing large number of isolates rapidly and directly from sputum samples makes Genotype MTBDRplus suitable as a first line screening assay for multidrug resistant tuberculosis. Most frequent mutation causing resistance is Serine-531-Leucine.