Setting : Twenty-nine epidemiologically unrelated and mostly multidrug-resistantMycobacterium tuberculosis (MDR-TB) strains from Peruvian patients.Objective : To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country.Design : Antimicrobial agent susceptibility testing, major genetic group designation, IS 6110 fingerprinting, spoligotyping, and automated deoxyribonucleic acid sequencing of regions of the katG, rpoB, embB, gyrA , and pncA genes with mutations commonly associated with drug resistance.Results : Nineteen isolates were found to be multidrug-resistant by susceptibility testing. IS6110 typing showed that virtually all isolates were unique and therefore had independently acquired drug resistance. Seventy-nine percent of isoniazid-resistant strains had a Ser315Thr amino acid change in KatG. Ninety-five percent of rifampin-resistant isolates had amino acid replacements in the rifampin-resistance determining region of RpoB. Six of 11 ethambutol-resistant strains had EmbB alterations. Eleven pyrazinamide-resistant strains had distinct mutations inpncA .Conclusion : Virtually all organisms evolved drug resistance independently. The types of drug resistance-associated mutations identified were very similar to changes occurring in isolates from other areas of the world. Nucleotide sequence-based strategies for rapid detection of drug resistance-conferring mutants will be applicable to organisms recovered in Peru, and potentially other areas of Latin America.