The peptides repertoire presented to CD8 T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP), which regulates thymus development, peripheral survival and function during lifetime of CD8 T cells. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by pancreatic β cells destruction mediated primarily by autoreactive CD8 T cells. Non-obese diabetic (NOD) mouse is an important animal model of T1D. Here, we deeply analyzed the MIP derived from NOD mice thymus and pancreas, and demonstrated that the thymus MIP source proteins partially shared with the MIP source proteins derived from NOD mice pancreas and β cell line. One H-2Kd restricted peptide SLC35B1 which was shared by MIP derived from both NOD mice pancreatic tissues and islet β-cell line, but absent in MIP from NOD thymus tissues, showed ability to stimulate IFN-γ secretion and proliferation of NOD mice splenic CD8 T cells. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a biological reference to identify potential autoantigens targeted by autoreactive CD8 T cells in T1D. Data are available via ProteomeXchange with identifier PXD031966. SIGNIFICANCE: The peptides repertoire presented to CD8 T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP). The MIP presented by thymic antigen presenting cells (APCs) is crucial for shaping CD8 T cell repertoire and self-tolerance, while the MIP presented by peripheral tissues and organs is not only involved in maintaining periphery CD8 T cell survival and homeostasis, but also mediates immune surveillance and autoimmune responses of CD8 T cells under pathological conditions. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by the destruction of pancreatic β cells, mediated primarily by autoreactive CD8 T cells. Non-obese diabetic (NOD) mouse is one of important animal models of spontaneous autoimmune diabetes that shares several key features with human T1D. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a good biological reference to identify potential autoantigens targeted by autoreactive CD8 T cells in T1D. It has great significance for further clarifying the immune recognition and effect mechanism of autoreactive CD8 T cells in the pathogenesis of T1D, and then developing antigen-specific immune intervention strategies.