Objective Mucous membrane pemphigoid is an immune-mediated subepithelial blistering disease consisting of immunologically heterogeneous subgroups. Differentiation between these subgroups is important because they differ in prognosis. This study uses oral mucosal pemphigoid specimens to investigate the utility of computer-aided fluorescence overlay antigen mapping and laser scanning confocal microscopy to differentiate subgroups of mucous membrane pemphigoid. Study design Thirty oral mucosal biopsy specimens were cryosectioned and immunostained, although only 13 could be analyzed due to technical difficulties. In vivo bound antibodies and molecular markers of the basement membrane zone were differentially labeled with fluorescent antibodies. Fluorescent signals were imaged, and the spatial localization of in vivo bound antibodies was compared with the markers and analyzed. Results In vivo bound IgG antibodies colocalized with β4-integrin in 3 cases, with laminin-5 in 8 cases, and with collagen VII in 2 cases. Conclusion Fluorescence overlay antigen mapping and laser scanning confocal microscopy are useful techniques to differentiate pemphigoid subgroups in oral biopsy specimens.