Abstract:  Desmogleins play critical roles in cell adhesion and skin blistering diseases, as they are the target antigens of autoimmune antibodies and bacterial toxins. We recently cloned several novel members of the desmoglein gene family, bringing the number of desmogleins to four in the rat and human genomes and six in the mouse. Here, we have produced a monoclonal antibody to a cytoplasmic epitope of Dsg4, assessed its specificity and compared it to several existing Dsg1–3 antibodies. We also demonstrated cross‐reactivity of commercially available and often used Dsg1 antibodies. Using these tools, we delineated the unique expression patterns of each desmoglein isoform in various human and mouse stratified squamous epithelia, including skin, hair, palm, and oral mucosa. Interestingly, in the epidermis, the expression of each desmoglein correlates with their gene arrangement in the cadherin locus. In human, Dsg4 was detected primarily in the granular and cornified cell layers of the epidermis, while present throughout all differentiated layers of the oral mucosa and palm, and in the matrix cells of anagen hair bulb. Similar pattern of expression for Dsg4 was observed in mouse, with the exception that it was expressed at significantly lower levels in the mouse epidermis. These results demonstrate the complexity of desmoglein gene expression and provide additional insights into the correlation between tissue expression patterns and disease phenotypes.