Pseudallescheria boydii (anamorph: Scedosporium apiospermum) and S. prolificans can cause severe infections in both the immunocompromized host and accidentally injured people. A species-specific polymerase chain reaction (PCR) enabling detection and discrimination of these fungi has not been available to date. In view of the difficult treatment especially of S. prolificans infections, a PCR-based detection system has been developed. Based on results of partial sequencing of ribosomal DNA, Scedosporium DNA could be amplified, either at the genus or at the species level. Hybridization probes for the identification of the PCR-products were established.