Systemic sclerosis is an autoimmune disease, clinically characterized by vascular and immune dysfunction, leading to fibrosis that can damage multiple organs. The presence of non-overlapping SSc-associated autoantibodies best presents the autoimmune nature of systemic sclerosis. The primary purpose of this study was to investigate the autoantibody profile in Iranian patients with systemic sclerosis. Sera from 481 patients with systemic sclerosis were collected from 2013 to 2016. The level of anti-nuclear antibodies (ANA) was quantitatively detected using the indirect immunofluorescence (IIF) method and the level of specific autoantibodies including anti-topoisomerase I antibody (ATA), anti-centromere antibody (ACA) and anti RNA polymerase III antibody (anti-RNAP III) were also determined qualitatively by the enzyme-linked immunosorbent assays (ELISA) technique. Among all patients evaluated, we found a predominance of females (86.7%) and 434 (90.2%) of patients showed positive ANA results by IIF. ANA was detected in 87.3% and 92.0% of limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) patients, respectively, which was not significantly different. The frequency of anti-RNAP III, ACA and ATA was 5.19%, 6.02% and 72.3%, respectively. Furthermore, anti-RNAP III, ATA and ANA levels were correlated with dcSSc, whereas ACA level was correlated with lcSSc. We confirmed that ATA expression is significantly higher in dcSSc patients. Our results showed that we have a lower frequency of ACA (6.02%) than most previous cohorts. The results of this study demonstrate that the clinical subtype of systemic sclerosis may correlate positively with the presence of specific autoantibodies.