The single nucleotide polymorphism (SNP) rs13266634 encodes either an Arginine (R) or a Tryptophan (W) (R325W) at the amino acid position 325 in the Zinc Transporter 8 (ZnT8) protein. Autoantibodies (Ab) that recognize ZnT8R, ZnT8W or both at the polymorphic site are common in newly diagnosed type 1 diabetes (T1D) patients. The epitope specificity and affinity of ZnT8Ab are poorly understood, but may be of importance for the prediction and clinical classification of T1D. Therefore, the aims were to 1) determine the immunogenicity of short (318331) ZnT8 peptides in mice and 2) test the affinity of short and long (268369) ZnT8 proteins in T1D patients positive for either ZnT8RAb or ZnT8wAb. Sera from BALB/cByJ mice immunized with short R, W or Q (Glutamine) ZnT8 peptides were tested for ZnT8-peptide antibodies in ELISA and radiobinding assay (RBA). Using reciprocal permutation experiment, short synthetic ZnT8R and ZnT8W (318331) and long in vitro transcription translation ZnT8R and ZnT8W (268369) proteins were tested in competitive RBA with R- and W-monospecific T1D sera samples. All mouse sera developed non-epitope-specific peptide antibodies in ELISA and only 6/12 mice had ZnT8-RWQ antibodies in RBA. Both long ZnT8R and ZnT8W (268369), but not any short, proteins displaced labelled ZnT8 (268369) proteins in binding to T1D ZnT8Ab-specific sera. The reciprocal cross-over tests showed that half-maximal displacement varied 2- to 11-fold indicating variable affinity of patient ZnT8Ab, signifying crucial autoantibody epitope spreading. The present approach should make it possible to dissect the importance of the R325W ZnT8 autoantigen epitope in the T1D pathogenesis.