Potential structural modifications of graphene exposed to gaseous tritium are important for membrane-based hydrogen isotope separation. Such modifications cannot be explained by electron irradiation alone. Instead, tritiation, caused by the tritium radicals remaining after the decay, is the primary effect causing the modification of the graphene surface, as confirmed by confocal Raman spectroscopy. The effect of the interaction of tritium atoms with the graphene surface exceeds that of electron irradiation at the average energy of the beta particles (5.7 keV). Compared to previously investigated high electron doses in the absence of tritium, remarkably low concentrations of tritium already induce a significant amount of sp3- and vacancy-type defects at short exposure times. Our findings are supported by molecular dynamics simulations of graphene bombardment with tritium atoms. As a consequence, tritium saturation of graphene may alter its permeability for hydrogen isotopes, thus affecting potential applications.