||Heavy-ion beam irradiation is one of the principal methods used to improve plant varieties. Research on mutagenic effects and molecular mechanisms of radiation is an important subject that is multi-disciplinary. However, it is impossible to fully characterize mutations induced by heavy-ion beam irradiation by results mainly focused on the phenotype, chromosome aberration, genetic polymorphism, or sequencing analysis of specific genes only. Here, we re-sequenced 11 mutagenesis progeny (M3) Arabidopsis lines derived from carbon-ion beam irradiation, and focused on substitutions and small insertion-deletion (INDELs). This revealed the molecular mutation spectrum and rate of carbon-ion beam irradiation on Arabidopsis at the genome wide level. This work can also inform theory and advances in radiation mutation breeding.