OBJECTIVE: To identify multiple infection events during anti-tuberculosis treatment in a cohort of pulmonary tuberculosis (PTB) patients in a local hospital in Callao, Peru. DESIGN: We evaluated 10 patients with smear-positive PTB who had two or more different isolates available during DOTS treatment. We determined drug susceptibility to rifampicin, isoniazid, ethambutol and streptomycin, and performed molecular sub-typing by restriction fragment length polymorphism (RFLP) and PCR. RESULTS: Drug susceptibility patterns changed from sensitive to resistant in the first and subsequent isolate of three of 10 patients. This change coincided with a dramatic alteration of their DNA fingerprint profile. We ruled out the possibility of cross-contamination or potential labelling errors. CONCLUSION: We found multiple infections with sensitive and resistant strains of Mycobacterium tuberculosis throughout the same treatment scheme in three patients with PTB. Further studies are needed to determine the frequency of exogenous re-infection and simultaneous/ mixed infection and their impact on TB control in Peru. © 2005 The Union.