Trichophyton tonsurans and T. equinum are two closely related sister species of dermatophytes, but differ in their preferred hosts, i.e., humans or horses, respectively. Routine procedures for their identification depend on studies of their phenotypic, physiological and biochemical characteristics, which are laborious and may yield ambiguous results. Molecular methods using rDNA ITS also had been judged to be insufficiently discriminatory. In the present study two genetic markers were sequenced in addition to the ITS region, i.e., partial β-tubulin (BT2) and translation elongation factor 1-α (TEF1). The TEF1 locus revealed a consistent differences in a 13 bp indel and an additional SNP between the two species, along with a single base substitution in BT2 and one ITS1, enabling unambiguous distinction of the two taxa. RFLP targeting the ITS region was evaluated as a potential tool for routine screening of suspected isolates of T. tonsurans and T. equinum.