We performed a comparative analysis of human and 12 non-human primates to identify sequence variations in known cancer genes. We identified 395 human-specific fixed non-silent substitutions that emerged during evolution of human. Using bioinformatics analyses for functional consequences, we identified a number of substitutions that are predicted to alter protein function; one of these mutations is located at the most evolutionarily conserved domain of human BRCA2. Display omitted •Hundreds of human-specific substitutions of cancer genes emerge during evolution•A substitution of BRCA2 codon 2662 is located at the most conserved domain•This substitution decreases the interactions of BRCA2 with DSS1•The substitution triggers a 20% reduction in recombinational DNA repair ability Huang et al. find a single base substitution in BRCA2 arose in the course of human evolution. This substitution (BRCA2M2662T) is fixed in the present-day human population. The substitution reduces BRCA2 activity and leads to reduced DNA repair by homologous recombination.