The 65-kDa glutamic acid decarboxylase (GAD65) autoantibodies (GAD65Abs), commonly found in type 1 diabetes mellitus (T1DM) patients, are also found at lower frequencies in type 2 diabetes mellitus (T2DM) patients. GAD65Abs in T1DM patients are epitope specific, in contrast to those found in other GAB65Ab-positive individuals, including T2DM patients. Our aim was to assess whether epitope-specific GAD65Abs, or the additional presence of islet antigen 2 (IA-2) autoantibodies, better define T1DM phenotypes among T2DM patients. GAD65 and IA-2 autoantibodies were analyzed in 1436 Sardinian subjects classified with T2DM and in 384 non-diabetic patient controls. Autoantibody binding specificity to the N-terminal, middle (M), and C-terminal (C) portions of the GAD65 molecule was evaluated. Among the T2DM patients, 5.1% had GAD65 (P < 0.001) and 2.4% had IA-2 autoantibodies, compared with 1.3 and 1.6%, respectively, among the controls. GAD65Ab-positive T2DM patients with M+C (epitope-specific) reactivity were found to have the lowest body mass index (P < 0.001), followed by GAB65Ab/LA-2Ab-positive patients (P < 0.01), andnon-M+C-reactive (non-epitope-specific) patients (P < 0.02). In GAD65Ab-positive T2DM patients, c-peptide levels were lower in M+C-reactive compared with non-M+C-reactive patients. Sardinian T2DM patients with M+C-predominant GAD65Ab reactivity have clinical features more similar to those of T1DM patients. Thus, GAD65Ab epitope analysis may help to define T1DM phenotypes among newly diagnosed GAD65Ab-positive patients classified with T2DM.