Updated publication reference for PubMed record(s): 30084848. The array of peptides presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC class I immunopeptidome. Although the MHC class I immunopeptidome is ubiquitous in mammals and represents a critical component of the adaptive immune system, very little is known about its baseline composition across all tissues and organs in vivo. In this study, we applied mass spectrometry (MS) technologies to draft a tissue-based atlas of the MHC class I immunopeptidome in the C56BL/6 mouse model. Overall, 19 normal mouse tissues were extracted and a total number of ~30,000 high-confidence H2Db/Kb-associated peptides were identified and annotated in the atlas. The raw MS output files, the catalogue of H2Db/Kb-associated peptides, the tissue origin(s) of individual peptides and the H2Db/Kb-specific peptide spectral libraries, which consist of consensus spectra calculated from repeat measurements of the same peptide sequence, were shared via the SysteMHC Atlas project (dataset identifier SYSMHC00018). We expect that this unique dataset will be further expended in the future and will be re-used by the community to 1) investigate the tissue-specificity of the immunopeptidome, 2) perform highly reproducible spectral library-based analysis of immunopeptidomes from mice cohorts, and 3) rapidly identify disease-specific H2Db/Kb peptide antigens from various mouse models of autoimmune diseases and cancers.