The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.