In patients with the major forms of epidermolysis bullosa simplex, either of the keratin genes KRT5 or KRT14 is mutated. This causes a disturbance of the filament network resulting in skin fragility and blistering. For KRT5, a genomic mutation detection system has been described previously. Mutation detection of KRT14 on a DNA level is, however, hampered by the presence of a highly homologous but nontranscribed KRT14 pseudogene. Consequently, mutation detection in epidermolysis bullosa simplex has mostly been carried out on cDNA synthesized from KRT5 and KRT14 transcripts in mRNA isolated from skin biopsies. Here we present a genomic mutation detection system for exons 1, 4, and 6 of KRT14 that encode the 1A, L1-2, and 2B domains of the keratin 14 protein containing the mutation hotspots. After cutting the KRT14 pseudogene genomic sequences with restriction enzymes while leaving the homologous genomic sequences of the functional gene intact, only the mutation hotspot-containing exons of the functional KRT14 gene are amplified. This is followed by direct sequencing of the polymerase chain reaction products. In this way, three novel mutations could be identified, Y415H, L419Q, and E422K, all located in the helix termination motif of the keratin 14 rod domain 2B, resulting in moderate, severe, and mild epidermolysis bullosa simplex phenotype, respectively. By obviating the need of KRT14 cDNA synthesis from RNA isolated from skin biopsies, this approach substantially facilitates the detection of KRT14 hotspot mutations.