The rapid identification of fungal pathogens in clinical specimens is a prerequisite for timely onset of the most appropriate treatment. The aim of the present study was to develop a sensitive and rapid method for the species-specific identification of clinically relevant fungi. We employed fluorescent polymerase chain reaction (PCR)-fragment length analysis of the highly variable internally transcribed spacer 2 (ITS2) region to identify individual fungal species by their specific amplicon sizes. The specificity of the technique was ascertained by the detailed analysis of 96 strains derived from 60 different human-pathogenic fungal species. To achieve adequate sensitivity for species identification in patients with invasive fungal infection, who often display very low pathogen loads in peripheral blood, the ITS2 region was amplified by semi-nested PCR prior to amplicon-length analysis. Serial specimens from 26 patients with documented fungal infections were investigated. The fungal pathogens identified included different Aspergillus and Candida species, Rhizopus oryzae and Fusarium oxysporum. Fragment length analysis of the ITS2 region upon amplification by semi-nested PCR permits the sensitive identification of fungal species. The technique can be readily implemented in routine diagnostics.