Genome sequencing has revealed a large number of shared and personal somatic mutations across human cancers. In principle, any genetic alteration affecting a protein-coding region has the potential to generate mutated peptides that are presented by surface HLA class I proteins that might be recognized by cytotoxic T cells. To test this possibility, we implemented a streamlined approach for the prediction and validation of such neoantigens derived from individual tumors and presented by patient-specific HLA alleles. We applied our computational pipeline to 91 chronic lymphocytic leukemias (CLLs) that underwent whole-exome sequencing (WES). We predicted ∼22 mutated HLA-binding peptides per leukemia (derived from ∼16 missense mutations) and experimentally confirmed HLA binding for ∼55% of such peptides. Two CLL patients that achieved long-term remission following allogeneic hematopoietic stem cell transplantation were monitored for CD8+ T-cell responses against predicted or confirmed HLA-binding peptides. Long-lived cytotoxic T-cell responses were detected against peptides generated from personal tumor mutations in ALMS1, C6ORF89, and FNDC3B presented on tumor cells. Finally, we applied our computational pipeline to WES data (N = 2488 samples) across 13 different cancer types and estimated dozens to thousands of predicted neoantigens per individual tumor, suggesting that neoantigens are frequent in most tumors. •Tumor neoantigens are a promising class of immunogens based on exquisite tumor specificity and the lack of central tolerance against them.•Massively parallel DNA sequencing with class I prediction enables systematic identification of tumor neoepitopes (including from CLL).