Sensitive screening for Aspergillus spp. using polymerase chain reaction (PCR) of whole blood samples in patients with haematological disorders has not been performed to date. In a 2-year study, 121 patients admitted to the University Hospital of Innsbruck for cancer chemotherapy without clinical signs of fungal infection were prospectively screened for Aspergillus spp. In 28 out of 121 (23%) patients, Aspergillus DNAaemia was detected. Of these patients, 16 (57%) were positive only once for Aspergillus DNA, but positivity was never associated with invasive aspergillosis. PCR positive episodes were short and resolved without antifungal treatment. Five patients (18%) had intermittent PCR positive results. Seven (25%) patients presented at least two consecutive positive PCR results; one of these patients developed invasive aspergillosis and another two were strongly suspected as having aspergillosis. Based on the criteria of the European Organization for Research and Treatment of Cancer case definitions, sensitivity and specificity of serial PCR monitoring were 75% and 96%. Positive PCR results became negative shortly after commencement of antifungal treatment, but the changes did not correlate with clinical responsiveness to treatment in three patients. Our results indicate the potential usefulness of PCR for screening for Aspergillus spp. in patients at risk, but without antifungal treatment.