Ionizing radiation induces clustered DNA damage, which contains localized multiple lesions in duplex DNA molecules. It has been thought that due to complex nature, clustered DNA damage is refractory to repair or associated with error-prone repair and results in severe biological endpoints as compared to sparsely-distributed lesions. High linear energy transfer (LET) radiations such as heavy ion beams exert a greater relative biological effect (RBE) than low LET radiations such as X- and gamma-rays. In the present study, we analyzed the yields of clustered DNA damage produced by high and low LET radiations using in vitro and in vivo systems and examined whether the differential formation of clustered DNA damage accounts for distinct RBE values of these radiations.