ABSTRACT Purpose and objectives Detection of drug resistance plays a crucial role in tuberculosis (TB) treatment and prevention of Mycobacterium tuberculosis (MTB) transmission. The aim of this study was to determine the levels and patterns of resistance of MTB isolates to two key anti-TB drugs (rifampicin, RIF and isoniazid, INH) and the type of mutations in drug resistance genes (rpoB, katG and inhA) of the isolates at the southern coastal region of Andhra Pradesh, India, using commercially available GenoType MTBDRplus assay under the Revised National TB Control Program. Methods GenoType MTBDRplus assay was performed on 2859 sputum smear-positive samples and the mutations in the genes responsible for resistance (rpoB, katG and inhA) were analyzed. Results Among the line probe assay (LPA) valid isolates (2894), 1990 (68.76%) were drug susceptible, 437 (15.13%) were INH monoresistant, 104 (3.59%) were RIF monoresistant, and 363 (12.54%) were multidrug resistant. Codon 531 of rpoB gene and codon 315 of katG gene were found to have the highest mutation frequency for RIF resistance (270/467; 57.81%) and INH resistance (501/800; 62.62%), respectively. The RIF resistant rpoB mutations observed in the samples were S531 L (57.81%), H526Y (8.56%), D516 V (6.42%), and H526D (6.20%). Mutations in inhA promoter were found in 24.75% INH resistant isolates with C15 T being the most common (85.85%). The turnaround times of the LPA test were from 48 to72 h. Conclusion The frequency of mutations in MTB in the coastal region of Andhra Pradesh, India, is similar to that in retreatment cases from most settings, with close to 80% in rpoB codon 516, 526, and 531, and over 80% in codons katG 315 and/or inhA promoter. The increase in INH monoresistance underlines the need for greater enforcement of national TB control programs.