Clinical associations of anti-SSA/Ro60 and anti-Ro52/TRIM21 antibodies are not yet fully established. In order to analyse the diagnostic utility of their separate detection, we retrospectively revised the clinical data of 200 anti-SSA/Ro60 and/or anti-Ro52/TRIM21 positive patients identified by line immunoassay during ANA routine detection. Anti-SSA/Ro60 positive patients showed a significantly higher prevalence of autoimmune diseases (AIDs) independently on the presence of anti-Ro52/TRIM21 (OR 3.13, 95% CI 1.10-8.88, p = 0.032). Anti-SSA/Ro60 was independently associated with systemic lupus erythematosus (SLE) when comparing with Sjögren's syndrome (SS) and other systemic AIDs (OR 3.46, 95% CI 1.08-11.06, p = 0.036). The more frequent specificity found in cutaneous lupus erythematosus (CLE) was also anti-SSA/Ro60. In contrast, detection of isolated anti-Ro52/TRIM21 was characteristic of SS (7/35, 20.0%), diffuse cutaneous systemic sclerosis (dcSSc) (3/4, 75.0%), primary biliary cirrhosis (PBC) (4/5, 80.0%) and, specially, of polymyositis/dermatomyositis (PM/DM) (6/6, 100%). In fact, anti-Ro52/TRIM21 was the only antibody detected in 4 out of the 6 PM/DM patients. Malignancies mainly account for the observed high prevalence of mono-specific anti-Ro52/TRIM21 in patients with non-AIDs (10/15, 62.5%). In conclusion, this retrospective study supports the routine distinction of anti-SSA/Ro60 and anti-Ro52/TRIM21 due to their different clinical associations.