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Objectives: This study aimed to assess the prevalence and clinical correlation of anti-RNA polymerase III antibodies (anti-RNAP III) in Chinese Han systemic sclerosis (SSc) patients. Methods: Serum samples from 236 patients with SSc, 125 patients with connective tissue diseases (CTD), and 166 healthy controls (HCs), recruited from Peking Union Medical College Hospital and 21 other medical centers in China, were tested for antibodies to RNA polymerase III by means of a line immunoassay (LIA) or an enzyme-linked immunosorbent assay (ELISA) kit. Results: Anti-RNAP III antibodies were found in 14/236 SSc patients (5.93%), 1/125 (0.80%) CTD patients, and 0/166 (0.00%) HCs. The prevalence of anti-RNAP III was higher in SSc patients than in the CTD and HC groups (p = 0.02, p = 0.001, respectively). Renal crisis was significantly more common in patients with anti-RNAP III than patients without anti-RNAP III (42.9 vs. 4.1%, p < 0.0001). Gastrointestinal involvement was significantly more common in patients without anti-RNAP III than patients with anti-RNAP III (53.6 vs. 21.4%, p = 0.039). There was good agreement between the ELISA and line immunoassay (LIA) detection capabilities for anti-RNAP III. Conclusions: The anti-RNAP III antibody, which was detected by ELISA, has diagnostic value for SSc and predictive value for SSc-related renal crisis. Both ELISA and LIA are very reliable methods for anti-RNAP III. Key Points • The prevalence of anti-RNAP III antibody was determined in Chinese SSc patients and performed ethnic differences. • The clinical association between anti-RNAP III antibody and Chinese SSc patients was evaluated in this research. • Methodological consistency of detection of anti-RNAP III antibody using commercial ELISA and LIA methods was evaluated in this research. [ABSTRACT FROM AUTHOR]