Invasive fungal infections (IFIs) are major complications after allogeneic hematopoietic SCT (HSCT). PCR-based assays able to detect fungal DNA have been reported to precede clinical diagnosis of IFI. We performed a prospective study to evaluate a PCR-based pre-emptive approach. Ninety-nine patients undergoing reduced-intensity conditioning (RIC) HSCT were followed with fungal PCR during the first 100 days post transplantation. Patients who tested positive were randomized to receive liposomal amphotericin B, or to no intervention. After day 100, PCR tests were performed only on clinical suspicion of IFI. A single positive PCR test was not associated with IFI, irrespective of treatment. After day 100, PCR tests for Aspergillus did not contribute to diagnosis of invasive aspergillosis (IA). The cumulative incidence rates of proven or probable IA during the first year after transplantation were 9%. GVHD grades II-IV (P=0.0014), CMV-seronegative recipient with CMV-seropositive donor (P0.001), and conditioning with alemtuzumab (P=0.014) were significant risk factors for developing IA in a multivariate model. In this study, PCR on peripheral blood was a poor indicator of IFI early after RIC HSCT. Aspergillus PCR tests performed on clinical suspicion after day 100 were insufficiently sensitive to be diagnostically useful.