Type 1 diabetes (T1D) is an autoimmune disorder in which the immune system destroys the insulin-producing beta cells of the pancreas. Autoreactive B cells that activate the immune response when recognizing self-proteins play an important, but ill-defined role in the progression of T1D. Autoreactive B cells that recognize any of three self-proteins related to T1D (Insulin, IA2, or GAD65) were isolated and RNA was sequenced for each cell from nondiabetic (ND), high risk, autoantibody positive prediabetic (AAB), and recently diagnosed T1D individuals. The frequency of autoreactive B cells found in AAB and T1D donors was increased, and these had altered gene expression in B cell signaling, antigen processing and presentation, autoimmunity, and pro-inflammatory pathways compared to ND donors. We found distinct B cell receptor heavy and light chain V gene usage in AAB and T1D donors from ND donors which were enriched for autoreactivity. Eighty-nine clonally expanded B cell receptor sequences were found across multiple donors and were shared almost entirely among AAB and T1D donors. Surprisingly, a substantial fraction of autoreactive B cell receptor sequences in AAB and T1D donors were found to be polyreactive. Polyreactivity was confirmed through ELISA analysis of selected recombinant monoclonal antibodies. These findings add evidence for unique autoreactive B cell activation and gene usage during T1D. Identification and further study of these autoreactive B cell receptor sequences can allow for their use as biomarkers of disease progression and potentially serve as targets for T1D treatment. The form and content of this abstract are approved. We recommend its publication.