Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in keratins K5 (keratin 5) and K14 (keratin 14), with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. Patients present with widely varying severity and are classified in three main subtypes: EBS Weber-Cockayne (EBS-WC), EBS Köbner (EBS-K), and EBS Dowling-Meara (EBS-DM), based on distribution and pattern of blisters. We could identify K5/K14 mutations in 20 out of the 43 families registered as affected by dominant EBS in Scotland; with previous studies this covers 70% of all Scottish EBS patients, making this the most comprehensively analyzed EBS population. Nine mutations are novel. All mutations lie within five previously identified rod domain hotspots and the severest blistering was associated with mutations in the helix boundary motifs. In some cases, the same mutation caused symptoms of EBS-WC and/or EBS-K, both within and between families, suggesting a contribution of additional factors to the phenotype. In some patients, no mutations were found in K5, K14, or K15, suggesting involvement of other genes. The results confirm that EBS is best considered as a single disorder with a spectrum of phenotypic variations, from severe EBS-DM at one extreme to mild EBS-WC at the other.