Biomarkers to stratify the complex and heterogeneous phenotypes of Sjogren’s syndrome (SS) are needed. We aimed to validate the prevalence of anti-aquaporin 5 (AQP5) IgG in a non-Korean cohort and to optimize the method to screen the anti-AQP5 IgG. The study cohort included 111 primary SS and 43 non-SS Sjögren’s International Collaborative Clinical Alliance (SICCA) controls that were obtained from the Sjögren’s International Collaborative Clinical Alliance registry, in addition to 35 systemic lupus erythematosus (SLE) and 35 rheumatoid arthritis (RA) phenotypes. Anti-AQP5 IgG was screened by cell-based immunofluorescence cytochemistry (CB-IFC) assay in the absence or presence of epitope peptides, as well as by ELISA using the epitope peptides as coated antigens. Anti-AQP5 IgG specific to an E1 epitope was best at discriminating between SS and non-SS, and the two different methods (CB-IFC and ELISA) presented comparable performance in diagnostic accuracy (0.690 vs. 0.707). Notably, the SLE and RA groups had substantially lower levels of anti-AQP5 IgG than the SS group. In addition, the presence of anti-AQP5_E1 IgG was associated with serologic and histopathological features of SS. In conclusion, a similar prevalence of anti-AQP5 IgG was confirmed in a non-Korean cohort. Screening anti-AQP5 autoantibodies may help to form subgroups of SS for targeted therapy.