To understand the pathogenicity and clinical significance of dermatophytes (also known as ringworms), the correct identification of these molds is essential. However, in routine practice they are notoriously difficult to classify and identify. The morphology of macroconidia, which are abundantly produced under suitable in vitro conditions, have provided useful criteria for the identification of many of the dermatophytes. However, several of them, including Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely produce macroconidia and cannot be easily identified. The objective of this study was to design, optimize, and evaluate real-time PCR as a tool for identifying dermatophytic fungi in a laboratory setting. The performance of the assay was evaluated using 64 dermatophyte isolates, i.e., 35 rare macroconidia-producing reference strains, including the six species mentioned above, and 29 clinical isolates from our laboratory, including M. canis (4), T. mentagrophytes (2), T. rubrum (20), T. rubrum with the 'raubitschekii' morphotype (2), and T. tonsurans (1). Real-time PCR correctly identified 10 taxonomically distinct dermatophytes, particularly rare macroconidia-producing species, with excellent sensitivity (100%). The advantages of the assay include the provision of accurate and reliable diagnoses of dermatophytic fungi.