Given the problems encountered in westernized countries with the laboratory diagnosis of malaria, namely sensitivity of the conventional methods and detection of mixed infections, a polymerase chain reaction (PCR)-based diagnosis has been developed and routinely used. The PCR used two sets of primers to simultaneously detect any infection due to the genus Plasmodium, or to the species P. falciparum. The PCR results were available within six hours. Five hundred twenty-nine patients were tested, of whom 136 were found positive by the PCR, and only 104 by the quantitative buffy coat (QBC) method. The 32 discrepancies were analyzed on the basis of the clinical data, and technical, molecular, and sequencing findings to ascertain the presence of Plasmodium DNA. The PCR-based diagnosis of malaria appeared to be a useful tool that was suitable as a second-line method when the results of conventional techniques were negative in patients presenting a syndromeconsistent with malaria, as well as yielding an accurate species identification.