Abstract: The reliability of PCR depends on many factors, such as high quality DNA pellets obtained from fixed Giemsa-stained blood samples, good reagents and adequate conditions of amplification. In account of this limitations, a retrospective epidemiological study for malaria diagnosis was conducted in a hypoendemic area, Sistan and Baluchestan province of south-east of Iran. For the first time this research designed to perform a nested PCR protocol using new and modified method of DNA extraction from negative Fixed and Giemsa Stained smears. This study demonstrated 11.55% PCR positive for Plasmodium vivax and Plasmodium falciparum which were undetectable by microscopy.