Background: The objective of this study was to determine the presence of anti-Dsg1 and Dsg3 antibodies in healthy subjects of the high Peruvian Amazon (Tuemal, Rodriguez de Mendoza province, department of Amazonas) to establish the theoretical presence of environmental factors or triggers in the area. Materials and methods: Cross-sectional study. The study population included persons of any age or gender, clinically healthy, who were evaluated by a dermatologist to confirm the absence of blistering diseases. Blood samples were analyzed by indirect immunofluorescence (IIF), immunoprecipitation (IP), anti-Dsg1 IgM antibody (Ab) enzyme-linked immunosorbent assay (ELISA), as well as anti-Dsg1 and anti-Dsg3 IgG Ab ELISA. Results: Participants included 21 healthy subjects comprised of 61.9% males and 38.1% females; 47.6% had a positive anti-Dsg1 Ab ELISA for total IgG (or any subclasses). IIF detected antibodies against intercellular spaces in one subject. Anti-Dsg1 Ab IP was mildly positive in 33.3% of the subjects. Anti-Dsg1 IgG subclasses found positive were: IgG1 (19.0%), IgG2 (33.3%), and IgG3 (28.6%); none of the samples were positive for anti-Dsg1 Ab IgM ELISA, and 23.8% of the subjects were positive for anti-Dsg3 Ab ELISA. The age distribution was similar for subjects positive for anti-Dsg1 and anti-Dsg3 Ab ELISA, with higher frequencies found among the 20–29 and 40–49 year-old age groups. Conclusion: A fraction of healthy subjects of the high Peruvian Amazon developed anti-Dsg1 and anti-Dsg3 antibodies, demonstrating the possible presence of environmental factors for endemic pemphigus (EP) at a higher altitude than previously described.