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Diagnosis of Pneumocystis jirovecii pneumonia (PCP) relies on lower respiratory tract sampling, often requiring bronchoscopy. Given its invasive nature, non-invasive diagnostic methods are desirable. In this retrospective case-control study, we evaluated the diagnostic performance of serum P. jirovecii PCR and β-D-glucan for PCP diagnosis. Adult patients with hematologic malignancies and positive P. jirovecii PCR in lower respiratory tract samples were included as cases and classified as PCP or colonization. Controls had negative BAL PCR. Stored serum samples were analyzed by P. jirovecii PCR and β-D-glucan. Forty-one cases (29 PCP, 12 colonization) and 36 controls were included. Serum P. jirovecii PCR was positive in 20/41 cases (49%) and in 15/29 patients with PCP (52%), while no controls were serum PCR-positive. For PCP diagnosis, serum PCR had a sensitivity of 52% and a specificity of 89%. For detection of P. jirovecii in the lower respiratory tract, sensitivity was 49% and specificity 100%. Detectable serum P. jirovecii DNA was associated with higher P. jirovecii load in respiratory tract samples and higher β-D-glucan levels, but not with the presence or severity of pneumonia. Serum P. jirovecii PCR is a highly specific marker of P. jirovecii in the lower respiratory tract and may serve as a useful initial non-invasive test in patients with suspected PCP, prior to invasive respiratory sampling.