Epidemiological data demonstrate a strong genetic influence on the etiology of thyroid autoimmune diseases (AITD) and T1 Diabetes (T1D). One candidate gene locus, that has been widely studied, is the HLA class II locus. Therefore, HLA class II is an obvious candidate gene-locus for the joint susceptibility to T1D and AITD. With the exception of a few reports, all studies showed a strong effect of HLA class II genes on the co-occurrence of T1D and AITD within families and in the same individual. Moreover, this association was consistent across different ethnic groups including Caucasians, Japanese, Koreans, and Chinese. Nevertheless, association studies of HLA in HT have been inconsistent, with different studies reporting association of HT with HLA-DR-3, -4, or -5. However, HLA-DR alleles are highly polymorphic and consist of many sequence variants. Therefore, we hypothesized that specific HLA-DR pocket sequence variants are associated with T1D and AITD when they occur in the same individual, with isolated HT, and that similar variants in the murine I-E locus predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. To test these hypotheses we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients, 105 T1D-AITD patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene (the murine homologue to human DR) in 22 strains of mice, 12 susceptible to EAT, and 10 resistant. In the T1D-AITD cohort of patients, at 11 of the 13 polymorphic positions were found amino acid residues strongly associated with the development of the disease (Tyr-26, Asp-28, Tyr-30, Tyr-37, Tyr-47, Leu-67, Gln-70, Lys-71, Arg-74, Asp-77 and Gly-86). Using logistic regression analysis, in the HT group, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71 that was strongly associated with HT (p=6.18x10-5, OR=3.73), with Lys-71 showing the strongest association as a single site (p=1.7x10-8, OR=2.98). This association was seen across HLA-DR types. In addition, the 5 amino acid haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 was also associated with HT (p=3.66x10-4, OR=3.45), and interestingly, Arg-74, while in tight linkage disequilibrium with Lys-71, contributed most to the DR pocket structure associated with HT. In mice the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were most strongly associated with EAT. Structural modeling studies, performed in the HT cohort of patients, demonstrated that pocket P4 was critical for the development of HT; it was more positively charged in HT patients than in controls. In mice both pockets, P1 and P4, influenced susceptibility to EAT. Surprisingly, the structure of the HT and EAT susceptible pockets were not similar. We conclude that: (1) Specific pocket amino acid signatures determine susceptibility to HT, T1D associated to AITD and EAT; (2) The specific amino acid signatures associated with HT and EAT cause significant structural changes in the MHC II peptide binding pocket, and may influence peptide binding, selectivity, and presentation in the initiation of thyroid autoimmunity in humans and in mice; (3) Since the HT- and EAT- associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with the initiation of HT and EAT.