Background <p>CD8⁺ T-cell activation is initiated by the recognition of epitopes presented on class I major histocompatibility complex (MHC-I) molecules. Identifying such epitopes is useful for molecular understanding of cellular immune responses and can guide the development of personalized vaccines for various diseases including cancer. For a few hundred common human and mouse MHC-I alleles, large datasets of ligands are available and machine learning MHC-I ligand predictors trained on such data reach high prediction accuracy. However, for the vast majority of other MHC-I alleles, no ligand is known.</p> Methods <p>We capitalize on an expanded architecture of our MHC-I ligand predictor (MixMHCpred3.0) to systematically assess the extent to which predictions of MHC-I ligands can be applied to MHC-I alleles that currently lack known ligand data.</p> Results <p>Our results reveal high prediction accuracy for most MHC-I alleles in human and in laboratory mouse strains, but significantly lower accuracy in other species. Our work further outlines some of the molecular determinants of MHC-I ligand prediction accuracy across alleles and species. Robust benchmarking on external data shows that our MHC-I ligand predictor demonstrates competitive performance relative to other state-of-the-art MHC-I ligand predictors and can be used for CD8⁺ T-cell epitope predictions.</p> Conclusions <p>Our work provides a valuable tool for predicting antigen presentation across all human and mouse MHC-I alleles. MixMHCpred3.0 tool is available at <a href="https://github.com/GfellerLab/MixMHCpred">https://github.com/GfellerLab/MixMHCpred</a>.</p>