A key to improving cancer immunotherapy will be the identification of tumor-specific ‘neo-antigens’ that arise from mutations and augmenting the resultant host immune response. In this study we identified single nucleotide variants (SNV) from RNA sequencing of asbestos induced murine mesothelioma cell lines, AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm the theoretical binding affinity of predicted peptides arising from high confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to twenty candidate mutation-carrying peptides of increased affinity plus corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumour bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neo-antigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non tumor bearing mice. The magnitude of the response to the Uqcrc2 neo-antigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neo-antigen. This work confirms that the approach taken, RNAseq plus peptide prediction and ELISPOT testing, is sufficient to identify natural tumor neo-antigens.