SETTING: National mycobacteriology reference laboratory in Peru conducting routine testing of susceptibility to isoniazid, rifampin, ethambutol, pyrazinamide, and streptomycin, in Mycobacterium tuberculosis isolates from previously treated patients. OBJECTIVE: To determine the percentage of isolates resistant to each of five anti-tuberculosis agents and to ascertain in these data the presence of trends of clinical relevance. DESIGN: Retrospective study of a national registry of M. tuberculosis isolates from patients referred for drug susceptibility testing between 1994 and 2001. RESULTS: Among 14736 isolates tested, 10837 (73.5%, 95%CI 72.8-74.3) demonstrated anti-tuberculosis resistance, and 8455 (57.4%, 95%CI 56.6-58.2) demonstrated resistance to at least both isoniazid and rifampin, by convention defined as multidrug-resistant tuberculosis (MDR-TB). A significant increasing trend could be discerned for resistance to each of the drugs tested and in isolates classified as MDR-TB (P < 0.001 for trend). Additional clinically relevant trends were found in polyresistance and multidrug resistance percentages. CONCLUSIONS: Data from a national reference laboratory can be used to inform the design of retreatment regimens.