Group I introns have the ability to catalyse their own excision (self-splice) from pre-RNA, and are found in a wide range of eukaryotic organisms. In fungal nuclear genomes, they have been identified in the small subunit (SSU) and large subunit (LSU) of the ribosomal RNA gene. Sequencing of the 3′ region of the LSU rRNA gene of the dermatophyte Trichophyton interdigitale revealed a 393 bp group I intron, Tin.2563, containing the four characteristic conserved motifs (P,Q,R and S) essential for self-splicing. The predicted secondary structure revealed nine sets of conserved paired regions (P1–P9), with most similarity to a subgroup IE intron of the entomopathogenic hyphomycete Beauveria bassiana. Tin.2563 was inserted at a site in the LSU rDNA corresponding to position 2563 of the Escherichia coli 23S rRNA. PCR and sequence analysis showed an intron to be present at an identical location in the LSU rDNA of many dermatophytes, although its distribution was erratic. In contrast, an intron was present at the same location in multiple isolates (n = 20) of the clinically important anthrophilic species Trichophyton rubrum and T. interdigitale. Conservation of intron insertion site, subgroup and P helix sequences showed intron genotyping to be unsuitable for strain identification in dermatophytes. Phylogenetic analysis of intron sequences from different dermatophyte species indicated that lateral transfer of the element was likely to be a rare event.