Microsporum ferrugineum, an uncommon causative agent of dermatophytosis, has restricted endemicity. Iranian strains suspected to be M. ferrugineum from two patients with tinea were analysed using the rDNA internal transcribed spacer (ITS) region and the partial β-tubulin (BT2) and translation elongation factor 1-α (TEF1) genes. Strains were compared to reference strains to differentiate M. ferrugineum from its relatives Microsporum canis and Microsporum audouinii. Inter-species differences for TEF1 and BT2 were found to be higher than for the ITS region, which is the current molecular standard for species identification in dermatophytes. Intra-species variation was zero for each of the markers. In silico analysis showed that the restriction enzymes BanI and BshNI were together sufficient to differentiate the three species based on TEF1, whereas a two-step digestion was needed with BT2 or the ITS region. The prevalence of M. ferrugineum in clinical samples in Iran appeared to be higher than suspected on the basis of routine phenotypic identification.