Background: The incidence of acquired resistance to antituberculous drugs of Mycobacterium tuberculosis in Chile is approximately 23%. Aim: To analyze the mutations associated with drug resistance in drug resistant strains of Mycobacterium tuberculosis. Material and Methods: In 28 drug resistant Mycobacterium tuberculosis strains isolated in Chile, genes leading to drug resistance were studied. DNA was amplifed by polymerase chain reaction (PCR) and sequencing was carried out using the ABI PRISM big dye terminator cycle sequencing ready reaction kit. Results: In rifampicin-resistant strains, the mutations in rpoβ gene were in the codons S531W/L (56%), D516Y (16%) and D516V (16%). The predominant mutation in katG gene was in the codon S315L (73%) in isoniazid-resistant strains. The mutation S95T was found in the 71% of ciprofoxacin resistant strains. Only one ethambutol resistant strain had the M306I mutation. Three unreported mutations in katG were identifed. Conclusions: Drug resistance associated mutations of Mycobacterium tuberculosis isolated in Chile were similar to those reported abroad.