Anti-p200 pemphigoid is an orphan subepidermal blistering disease characterized by autoantibody binding to the dermal site of human salt-split skin (SSS) and reactivity against a 200 kDa protein in the extract of human dermis by immunoblotting. Here, we aimed at the thorough serological characterization of anti-p200 pemphigoid. This retrospective study included all 243 patients with anti-p200 pemphigoid diagnosed between 2008 and 2017 in our routine autoimmune laboratory. Patients were diagnosed based on a compatible clinical picture and reactivity with the p200 protein in dermal extract by immunoblotting as described previously. In addition, all sera were subjected to indirect immunofluorescence microscopy on SSS and immunoblotting with the extracellular matrix of cultured keratinocytes for reactivity against laminin 332 and ELISA or immunoblotting applying the recombinant NC1 domain of collagen type VII. Autoantibodies against the dermal side of SSS were found in 91% (221/243) of patients and against the p200 protein and laminin γ1 in 100% (243/243) and 88% (209/238) of sera, respectively. IgG reactivity to laminin 332 was observed in 21% (48/233) of patients, in 98% of those (47/48) against the α3 chain of laminin 332. IgG autoantibodies to type VII collagen were detected in 2% (4/243) of patients. 30% (73/243) of sera showed additional reactivity to the epidermal site of SSS. In 58% (42/73) of epidermal binding sera, antibodies against BP180 or BP230 were detected by ELISA, while in 42% (31/73) of these sera, the target antigen remained unclear. In 37% (89/243) of sera, reactivity against one antigen in addition to p200/laminin γ1 and in 7% (16/243) against 2 additional antigens was detected. In summary, in this so far largest cohort of anti-p200 pemphigoid patients, epitope spreading was a common phenomenon and most frequently included laminin 332 followed by BP180, BP230, and type VII collagen.