Pneumocystis jirovecii is a common cause of life-threatening pneumonia among immunocompromised patients. Since P. jirovecii cannot be cultured, specific identification of it depends on examining respiratory specimens. In the last decade, PCR has been developed which allows the detection of very low levels of P. jirovecii not detectable by routine histochemical staining. We have shown that the direct immunofluorescence assay can be replaced by a real-time PCR assay given its feasibility, sensitivity, and specificity, for the detection of P. jirovecii. A negative PCR, performed on a LightCycler System(®), enables a diagnosis of Pneumocystis jirovecii pneumonia (PjP) to be excluded, and the semiquantitative result with the application of some cutoff values can have a role in distinguishing between colonized or subclinically infected patients and PjP patients.