Abstract: Heavy-ion irradiation mutation breeding is a promising approach for food germplasm innovation and has attracted widespread attention and application. To enhance the breeding applications of heavy ion radiation, it is necessary to further investigate the mechanisms of repair and mutation evolution of DNA clustered damage induced by heavy ion irradiation in plants, based on the ability to localize the heavy-ion irradiation targets at the DNA sequence level. Currently, although some conventional reporter gene-based methods have been established to localize heavy ion radiation targets, their efficiency remains suboptimal. In this work, we propose a new method to accurately localize the targeted loci of heavy-ion irradiation on DNA molecules in Arabidopsis thaliana, based on the abundance of DNA double-strand breaks (DSB) in the core of clustered DNA damage and the specific response of the homologous recombination mechanism to DSB, and its feasibility was initially determined by comparing the mutagenic distribution characteristics of γ and carbon ion radiation. To further improve the localization efficiency, by linking the lacI screening gene to the flanking sequence of the HR-GUS gene, we found that the characteristics of heavy-ion irradiation mutagenesis were fragment deletion and multi-loci mutation. This experimental system will provide a good technical support for further elucidating the mechanisms of heavy ion mutagenesis.