The genetic diversity of the emerging fungal pathogen Trichoderma longibrachiatum was examined at the level of mitochondrial DNA. The 17 investigated strains, comprising nine clinical and eight non-clinical isolates, exhibited seven and ten different mitochondrial DNA profiles by using the restriction enzymes BsuRI and Hin6I, respectively. The sizes of mitochondrial DNAs varied from 34.9 to 39.5 kb. The discriminatory power of the method was higher than that of internal transcribed spacer sequence analysis and therefore should be more suitable for identification and epidemiological investigations. However, clinical and non-clinical isolates did not form separate clusters on the resulting dendrogram and thus there was no indication of a correlation between genetic structure and pathogenicity of the isolates.