Dermatophytes, related to Trichophyton, Microsporum and Epidermophyton genera, are the main causative agents of superficial mycosis affected scalp, smooth skin and nail plates. KOH-microscopy and mycological culture are the basic methods for diagnosis of dermatomycosis. The major drawbacks of these methods are their low sensitivity, against the background of impossibility of microscopic species identification, which is important for diagnosis and appropriate treatment, as well as long duration of cultural examination. Real-Time polymerase chain reaction (RT-PCR) is increasingly being used abroad as an alternative approach, which has high analytical and diagnostic characteristics, combined with short duration of examination and ability to identify genus and species. The first set of RT-PCR based reagents in the Russian Federation allows to identify DNA of T. rubrum, T. mentagrophytes complex, T. tonsurans, M. canis, E. Floccosum in biological materials — skin and its appendages. The efficacy of this set application compared to KOH-microscopy and mycological culture in samples of skin (154), hair (62) and nail plates (42), received from 258 patients, was evaluated. RT-qPCR revealed 88 (34%) cases of dermatophytosis, while with mycological culture 32 (12%) cases were detected, and with microscopy — 26 (10%) cases. Detection rate of M. canis by RT-PCR method was 1.8 and 1.3 times higher than by mycological culture in skin and hair samples relatively. Detection rate of T. tonsurans by RT-PCR method was 2.7 times higher in skin samples and 6 times higher in hair samples than by mycological culture. T. mentagrophytes in samples of skin and nails, as well as T. rubrum in skin samples were detected only by RT-PCR method. Detection rate of T. rubrum in nail samples by the RT-PCR method was 16 times higher compared to mycological culture and 5.3 times higher compared to microscopy. The introduction of RT-PCR largely solves the issues of sensitivity, specificity and duration of results obtaining in the diagnosis of dermatophytosis.