Human leukocyte antigens (HLAs) are encoded by the most polymorphic genes in the human genome. HLA class I alleles control antigen presentation for T cell recognition, which is pivotal for autoimmunity, infectious diseases and cancer. Current knowledge of HLA-bound peptides is limited, skewed and falls short of population-wide HLA binding profiles for high-value targets. Here we present ESCAPE-seq (enhanced single-chain antigen presentation sequencing), a massively parallel platform for comprehensive screening of class I HLA-peptide combinations for antigen presentation via deep DNA sequencing. ESCAPE-seq demonstrates programmability, high throughput, sensitivity and nominated viral and cancer epitopes. We simultaneously assessed over 75,000 peptide-HLA combinations, revealing broadly presented epitopes from oncogenic driver mutations and fusions across diverse HLA-A, HLA-B and HLA-C alleles that cover 90% of the human population. We further identified epitopes that are differentially presented, comparing oncogenic hotspot mutations versus wild type. ESCAPE-seq enables one-shot population-wide antigen presentation discovery, offering insights into HLA specificity and immune recognition of genomic mutations.