Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru. Twenty-nine epidemiological unrelated and mostly multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains from Peruvian patients. To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country. Antimicrobial agent susceptibility testing, major genetic group designation, IS6110 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. 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 in pncA. 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.