Background: Dystrophic Epidermolysis bullosa (DEB) is one of the EB types caused by a mutation in the COL7A1 gene, which encodes collagen type 7 and is the main component of anchoring fibrils. Objectives: This study aimed to investigate COL7A1 gene mutations in five families of patients. Khuzestan is suffering from EB. Methods: After extracting genomic DNA from peripheral blood samples of probands, COL7A1 gene screening was performed using polymerase chain reaction and direct sequencing of exons 36-37 and their adjacent introns with the Sanger sequencing method. Results: The identified mutations included a new homozygous deletion mutation in exon 36 (c.2669delC) that resulted in a change in the open reading frame. Other obtained variants are the common mutations c.4233delC and c.4233delT in exon 37, which were identified as proline substitution mutations in one mutant allele in the homozygous form and three allele mutants in the heterozygous form. Conclusions: In this study, presenting a new mutation in the COL7A1 gene is the focus of the molecular heterogeneity of DEB. In addition, the efficient identification of COL7A1 gene mutations provides more information for the accurate diagnosis and prediction of the disease, genetic counseling, and prenatal diagnosis.