We compared light microscopy examination and a semi-nested multiplex PCR (SnM-PCR) assay in endemic areas of the Islamic Republic of Iran. A total of 68 individuals with malaria-positive and suspected malaria symptoms were included in the study. Giemsa-stained thick blood films were examined under a light microscope for malaria parasites in 100 and 200 fields. DNA was extracted from blood samples and SnMPCR based on the amplification of the small sub-unit ribosomal RNA (ssrRNA) gene sequences was applied. Microscopical examination showed that 48.5% (33.8% P. vivax and 14.7% P. falciparum) and 50% (35.3% P. vivax and 14.7% P. falciparum) of the samples were positive in 100 and 200 fields respectively. SnM-PCR showed the same results as the 200 field microscopy.