Background: Use of Pneumocystis jirovecii (Pj) polymerase chain reaction (PCR) is limited by Pj colonization causing lower specificity. We conducted a retrospective, clinical evaluation of patients who were tested positive for Pj PCR in either bronchoalveolar lavage or bronchial washing fluid. Methods: Between May 2009 and October 2011, Pj PCR was performed in 961 adult patients. We defined definitive PCP as the trimethoprim-sulfamethoxazole treatment group. Results: One hundred twenty one patients (12.6%) with a positive Pj PCR result were enrolled. 57 patients were identified as a definitive PCP group (47.1%) and 64 patients were in the non-treatment group (52.9%). The underlying conditions included HIV (n=6, 5%), hematological malignancies (n=35, 28.9%), solid organ transplantation (n=10, 8.3%), solid tumors (n=29, 24%), autoimmune diseases (n=13, 10.7%), and others (n=28, 23.1%). The overall mortality rate for patients in the definitive PCP group was 38.6% (n=22). Univariable analysis showed that age, underlying conditions, use of systemic steroid, and dyspnea differed significantly between two groups. Independent risk factors for definitive PCP were underlying conditions- HIV, hematological malignancies, or solid organ transplantation (odds ratio [OR], 5.904; P=0.000), and systemic use of steroids (OR, 2.666; P=0.049). Conclusions: This study shows that patients with HIV, hematological malignancies or solid organ transplantation, and with systemic steroid use display a higher risk of definitive PCP when tested positive for Pj PCR.