Epidermolysis bullosa (EB) comprises a diverse group of rare genetic skin fragility disorders. Accurate diagnosis and genetic counseling necessitate genetic analysis. This is the largest genetic study of EB in Brazil and one of the largest worldwide.To characterize the genetic basis of EB in this large, diverse cohort and provide a comprehensive overview of the mutational landscape in this population.Whole-exome sequencing was performed on 393 individuals from 349 families with a clinical or suspected EB diagnosis.A definitive EB diagnosis or differential diagnosis was achieved in 91.86% of our sample, including one case of Peeling Skin Syndrome 2. We identified 211 pathogenic or likely pathogenic variants in 12 genes, including 63 novel variants. Dystrophic EB was the most prevalent type (74.17%), with 149 variants identified (46 novel). The most frequent variant was the COL7A1 c.6527_6528insC. EB simplex accounted for 18.61%, with 36 variants identified (9 novel). The most frequent variants affected residue 125 of KRT14 . Junctional EB comprised 6.67%, with 22 variants (8 novel) identified. The LAMB3 c.31dup variant was the most prevalent. Kindler EB comprised 0.55% of the patients, and four variants were identified. For 163 individuals, the initial clinical diagnosis differed from the genetically confirmed EB type, highlighting the importance of genetic diagnosis.This study provides a comprehensive genetic overview of EB in Brazil, highlighting EB type prevalence and novel variants, crucial for genetic counseling, clinical management, and targeted therapies, potentially benefiting patients globally.