Fusarium species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA ofFusarium species. The DNAs of the main Fusariumspecies and Neocosmospora vasinfecta but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of Fusarium solani DNA by ethidium bromide staining. To test the ability of this PCR system to detect Fusarium DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected Fusarium DNA in mouse tissues and in spiked human blood. Furthermore, F. solani, Fusarium moniliforme, and Fusarium oxysporum were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or severalFusarium species. The method developed may be useful for the rapid detection and identification of Fusarium species both from culture and from clinical samples.