The emergence of drug-resistant tuberculosis is a major global public health threat. Thailand is one of the top 14 countries with high tuberculosis, tuberculosis co-infected with Human Immunodeficiency Virus (HIV) and multi-drug resistant tuberculosis rates. Immediate detection of drug-resistant tuberculosis is necessary to reduce mortality and morbidity by effectively providing treatment to ameliorate the formation of resistant strains. Limited data exist of mutation profiles in Northeastern Thailand. Here, 65 drug-resistant Mycobacterium tuberculosis isolates were used to detect mutations by polymerase chain reaction (PCR) and DNA sequencing. In the katG gene, mutations occurred in 47(79.7%) among 59 isoniazid resistant samples. For rpoB gene, 31 (96.9%) were observed as mutations in 32 rifampicin resistant isolates. Of 47 katG mutation samples, 45 (95.7%) had mutations in katG 315 codon and 2 (4.3%) showed novel mutations at katG 365 with amino acid substitution of CCG-CGG (Pro-Arg). Moreover, 18 (58.1%) mutations at rpoB 531, 11 (35.5%) mutations at rpoB 516, 10 (32.3%) mutations at rpoB 526 and 1 (3.2%) mutation at rpoB 533 were found between 31 rifampicin resistant samples. Common and novel mutations of the rpoB and katG genes in Northeastern Thailand were generated. DNA sequencing showed high accuracy, while conventional polymerase chain reaction could be applied as an initial marker for screening rifampicin and isoniazid drug-resistant Mycobacterium tuberculosis in Northeastern Thailand. Mutations reported here should be considered when developing new molecular diagnostic methods for implementation in Northeastern Thailand.