Mycoplasma pneumoniae is a causative agent of human respiratory tract infection of which the clinical features are not significantly different from those of infections caused by other respiratory pathogens. The diagnosis is based principally on laboratory tests. Since conventional methods such as culture and serological tests are time-consuming, insensitive, and non-specific, polymerase chain reaction (PCR) was employed for laboratory diagnostics. This study was aimed to develop PCR method to detect M. pneumoniae by designing primers to amplify fragment of the P1 adhesin gene. Two protocols, PCR-probe hybridization and nested PCR, were carried out. False-positive result due to amplicon carry over was prevented by using dUTP instead of dTTP and the addition of enzyme uracil DNA glycosylase (UDG). For nested PCR, UDG was added only in the first round reaction mixture. The sensitivity of PCR was 10 fg of M, pneumoniae DNA as detected by agarose gel electrophoresis and increased to be 1 fg as detected by either probe hybridization or nested PCR. The specificity of PCR was tested with DNAs from Mycoplasma spp, a variety of different bacterial genera and human leukocyte. All gave negative results. Considering of the speed, sensitivity, specificity and the prevention of amplicon carryover, the developed PCR-based protocols were suitable and reliable for the detection of M. pneumoniae in routine laboratory.