We used real-time polymerase chain reaction (PCR) targeting the cdc2 gene and direct fluorescent microscopy examination (DFME) to evaluate the prevalence of Pneumocystis jirovecii among immunocompetent patients without clinical pulmonary infection and immunosuppressed patients evaluated for opportunistic pulmonary infections. Among 102 bronchoalveolar lavage samples collected from immunocompetent patients without infection, none tested positive for P. jirovecii by either DFME or real-time PCR despite the presence of other comorbidities. Among patients with suspected pulmonary infection and tested with either assay, real-time PCR produced a higher number of positive results compared to DFME and increased P. jirovecii detection by 7% when added to DFME-negative samples. Real-time PCR may have increased sensitivity for P. jirovecii detection over DFME and decrease the risk of sample contamination compared to conventional and nested PCR. The use of single-copy gene targets (e.g., cdc2) may lower the rate of "colonization" detection and confer a high predictive value for Pneumocystis pneumonia.