Junctional Epidermolysis Bullosa (EB) is a severe congenital disease affecting the skin at the extremities of the limbs and the mucous membranes. In sheep, this recessive disease causes perinatal death of affected lambs. Multiple observations of EB cases were recently reported in the French Mouton Vendéen meat sheep breed. Skin biopsies of 6 affected lambs and when available, blood samples from the parents (n= 7) and unaffected full-sib lambs (n = 4) were collected for genomic DNA extraction. From a bibliography study and analysis of the OMIM database (Online Mendelian of Human Genes and Genetic Disorders), we have focused on 12 candidate genes mainly belonging to the collagen, laminin, integrin and keratin families. Based on the hypothesis of a recessive transmission of a deleterious variant, we have performed whole-genome sequencing of 2 unrelated EB-affected lambs (supposed homozygous carriers) and 1 unaffected full-sib (supposed heterozygous or non-carrier). Using the GATK workflow on a Galaxy platform, we have identified a novel SNP in the exon 23 of the ITGB4 gene of the integrin family (OAR11_v4.0, g. 54799925 G > A (p.885 R > *)) whose variant allele causes a premature stop codon. By a specific RFLP assay, we have determined that all EB-affected lambs were homozygous for this variant allele, their parents were heterozygous and the full-sibs were either heterozygous or non-carrier, fitting well with the working hypothesis. Following this primary discovery, a larger set of Mouton Vendéen animals was genotyped. We estimated the population allele frequency at 6.8% by genotyping a cohort of renewal ewe lambs (n = 1227). We also found a 5.7% allele frequency among the breeding rams present in 2018 (n = 1007) in artificial insemination center, progeny-testing station, and for natural mating in farms. Moreover, the specific genotyping of producing ewes in the most EB-affected flocks revealed a variant allele frequency up to 13.3%, due to overuse of inbreeding strategy. In conclusion, the discovery of a new mutation in ITGB4 causing EB in sheep will improve the selection scheme management of the Mouton Vendéen breed to limit the dissemination of this disease.