Abstract: Desmosomes are major intercellular adhesion junctions that provide stable cell–cell contacts and mechanical strength to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells. Desmogleins (Dsg) are transmembrane core components of the desmosomes, and belong to the cadherin supergene family of calcium‐dependent adhesion molecules. Currently, there are three known isoforms of Dsgs (Dsg1, Dsg2, and Dsg3), encoded by distinct genes that are differentially expressed to determine their tissue specificity and differentiation state of epithelial cells. In this study, we cloned a novel mouse desmoglein gene sharing high homology to both mouse and human Dsg1. We propose to designate the previously published mouse Dsg1 gene as Dsg1‐α and the new gene as Dsg1‐β. Analysis of intron/exon organization of the Dsg1‐α and Dsg1‐β genes revealed significant conservation. The full‐length mouse Dsg1‐β cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids. Dsg1‐β protein shares 94% and 76% identity with mouse Dsg1‐α and human DSG1, respectively. RT‐PCR using a multitissue cDNA panel demonstrated that while Dsg1‐α mRNA was expressed in 15‐ to 17‐day‐old embryos and adult spleen and testis, Dsg1‐β mRNA was detected in 17‐day‐old embryos only. To assess subcellular localization, a FLAG‐tagged expression construct of Dsg1‐β was transiently expressed in epithelial HaCaT cells. Dsg1‐β‐FLAG was found at the cell–cell border and was recognized by the anti‐Dsg1/Dsg2 antibody DG3.10. In summary, we have cloned and characterized a novel member of the mouse desmoglein gene family, Dsg1‐β.