Objective To analyze the pathogenic gene mutation characteristics in three families with epidermolysis bullosa simplex (EBS) and explore the association between genotype and phenotype, providing evidence for molecular diagnosis and genetic counseling of EBS. Methods Three EBS families admitted between 2019 and 2023 were enrolled. Whole-exome sequencing (WES) was performed to screen candidate pathogenic mutations, followed by Sanger sequencing validation. For the novel KRT5 mutation, protein structure modeling and functional prediction were conducted using AlphaFold 3 and PDBePISA. Individualized intervention plans were formulated and followed up. Results All patients in the three families met the diagnostic criteria for localized EBS (EBS-loc), and there were differences in the extent, severity, and disease course. Genetic testing revealed that family 1 carried a novel missense mutation in the KRT5 gene (NM_000424.4): c.1291A>G (p.Lys431Glu), which was an autosomal dominant inheritance; family 2 carried a known missense mutation in the KRT14 gene (NM_000526.5): c.374G>A (p.Arg125His), which was co-segregated with the phenotype; family 3 carried a novel missense mutation in the KRT5 gene (NM_000424.4): c.428T>A (p.Val143Asp). Protein structure prediction showed that the p.Lys431Glu mutation would weaken the binding affinity and structural stability of the KRT5-KRT14 heterodimer. Conclusion This study identified a novel KRT5 (NM_000424.4): c.1291A>G (p.Lys431Glu) mutation that has not been reported before, enriching the EBS mutation spectrum. Protein structure prediction revealed the pathogenic mechanism of this mutation. Individualized comprehensive management can significantly improve the prognosis of EBS patients, and genetic testing provides an important basis for clear diagnosis, genetic counseling, and individualized management.