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Over the past decade, anti-malarial drug resistance has rapidly become a major public health problem in the Peruvian Amazon. This study compared polymerase chain reaction (PCR) to light microscopy for diagnosing and monitoring the parasitological response of malaria patients to anti-malarial chemotherapy in the Peruvian Amazon region of Iquitos. Typing of P. falciparum using MSP1, MSP2, and glutamine-rich protein distinguished among infecting parasites. Most (73%) P. falciparum patients were parasitologically resistant to sulfadoxine-pyrimethamine (RI = 10, RII = 1). Sensitivity of microscopy was lower than PCR (69% for P. vivax and 78% for P. falciparum), but parasite clearance times were comparable between microscopy and PCR. PCR sensitively and specifically detected mixed infections and low-level parasitemia indicative of drug resistance, making this approach of practical use for the control of malaria at the public health level. Genotyping malaria parasites will be useful to distinguish drug failure from new infections in clinical trials of anti-malarial drugs in the Peruvian Amazon region.