We evaluated the utility of Aspergillus PCR as a tool in diagnosing invasive aspergillosis in patients at risk. Aspergillosis was assessed according to the European Organization for Research and Treatment of Cancer (EORTC), Mycosis Study Group definitions. Nine and seven patients with proven and probable aspergillosis respectively were evaluated. Whole blood samples prior (n = 41) and during antifungal treatment (n = 67), and tissue specimens (n = 9) and/or bronchoalveolar lavage fluids (n = 7) were investigated. In patients with proven infections, the sensitivities of PCR of lung samples were 100%, of blood samples prior treatment were 66%, and during treatment 55%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in two of four patients with proven infection. Consecutive positive PCR results for Aspergillus are fatal as two of five patients died. In patients with probable infections, the sensitivities of PCR of lung fluids were 85%, of blood samples prior treatment were 57%, and during treatment 42%. The benefits of PCR diagnosing and screening of whole blood are limited if sampling takes place once treatment has started. The performance of Aspergillus PCR from tissue samples should be recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.