Microsporum canis, for which the natural hosts are cats and dogs, is the most prevalent zoophilic agent causing tinea capitis and tinea corporis in humans. We present here a diagnostic PCR test for M. canis, since its detection and species identification is relevant to the choice of treatment and to the understanding of a probable source of infection. An M. canis-specific PCR was evaluated using 130 clinical isolates of dermatophytes (including M. canis [n = 15] and 13 other species), 10 yeast or mold isolates, 12 hair and skin samples from animals with or without experimental M. canis infection, and 35 patient specimens, including seven specimens positive for M. canis and 15 dermatophyte negative samples. All pure cultures, animal specimens and clinical samples with M. canis were detected by the PCR test, whereas none of the other fungal isolates or samples without M. canis was negative. This study indicates that the PCR test for M. canis identification applied directly to patient specimens or animal hair, as well as to clinical isolates had 100% specificity and sensitivity.