Summary Anti-Ro60 is one of the most common and clinically important serum autoantibodies that has a number of diagnostic and predictive capabilities. Most diagnostic laboratories report this simply as a qualitative positive/negative result. The objective of this study was to examine the clinical and serological relevance of a novel subset of anti-Ro60 in patients who display low levels of anti-Ro60 (anti-Ro60low). We retrospectively identified anti-Ro60 sera during a 12-month period at a major immunopathology diagnostic laboratory in Australia. These all were anti-Ro60-precipitin-positive on the diagnostic gold standard counter-immuno-electrophoresis (CIEP). Lineblot immunoassay was used to stratify patients into either anti-Ro60low or anti-Ro60high subsets. We compared the medical and laboratory parameters associated with each group. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry techniques were used to analyse the serological and molecular basis behind the two subsets. Anti-Ro60low patients displayed less serological activity than anti-Ro60high patients with less intermolecular spreading, hypergammaglobulinaemia and less tendency to undergo anti-Ro60 isotype-switching than anti-Ro60high patients. Mass spectrometric typing of the anti-Ro60low subset showed restricted variable heavy chain subfamily usage and amino acid point mutations. This subset also displayed clinical relevance, being present in a number of patients with systemic autoimmune rheumatic diseases (SARD). We identify a novel anti-Ro60low patient subset that is distinct from anti-Ro60high patients serologically and molecularly. It is not clear whether they arise from common or separate origins; however, they probably have different developmental pathways to account for the stark difference in immunological maturity. We hence demonstrate significance to anti-Ro60low and justify accurate detection in the diagnostic laboratory.