Approximately one third of community acquired pneumonia cases are caused by atypical pneumonia agents, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophila pneumoniae (formerly Chlamydia pneumoniae). The laboratory diagnosis of these organisms is difficult and time-consuming by conventional microbiological techniques. Polymerase chain reaction (PCR) is one of the important tools which can circumvent this problem. A multiplex PCR assay was developed to achieve the diagnosis of these three organisms in a single tube. Primers used in PCR were selected in a way that they amplified different length DNA fragments from different agents but they all worked at the same amplification conditions. Therefore the organisms could be diagnosed according to the length of amplified products by agarose gel electrophoresis without using any hybridization probes. After development of the multiplex PCR method, totally 309 clinical samples which were sent to our laboratory for single-agent PCR, were also evaluated by this technique. The results showed that the multiplex PCR assay is a sensitive, useful, cheap, and rapid diagnostic tool for the management of pneumonia patients.