Background: Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection in HIV-positive and also in immunocompromised HIV-negative patients. PCR analysis of pulmonary samples has become an essential element in PCP laboratory diagnosis. Currently, many commercially PCR-based tests are available for P jirovecii detection and need to be evaluated. Objectives: We evaluated the performance of the RealStar® P jirovecii PCR kit for PCP diagnosis. Methods: We performed the RealStar® P jirovecii PCR and an in-house PCR in 219 pulmonary samples. We then assessed the performance of the RealStar® P jirovecii PCR kit by classifying patients in proven, probable, possible PCP or no final diagnosis, on the basis of the clinical and radiological signs and direct examination of bronchoalveolar lavage samples. Results: The results showed excellent concordance (96.8%) with another in-house PCR, previously used in the laboratory. The available clinical data allowed classifying 219 patients as having proven PCP (n = 6), probable PCP (n = 27), possible PCP (n = 29) and no final diagnosis of PCP (n = 157). The RealStar® P jirovecii PCR kit performed well with samples from patients with proven and probable PCP, as indicated by the detection of P jirovecii DNA in all these samples. The percentage of positive samples in the possible PCP category was 75.9%. In patients with no final diagnosis of PCP, P jirovecii DNA was detected in 13.4% of samples, indicating colonisation by this pathogen. Conclusions: The RealStar® P jirovecii PCR kit shows excellent performance for PCP diagnosis.