We previously showed that moss (Physcomitrium patens) cells are highly radioresistant and suggested that P. patens uses an efficient mechanism to repair DNA double-strand breaks (DSBs). Homologous recombination (HR), canonical non-homologous end-joining, and alternative end-joining are the major pathways used to repair DSBs. To identify the DSB repair pathway used in P. patens, we generated knockout (KO) plants for LIG4, POLQ, and RAD51B, which play major roles in canonical non-homologous end-joining, alternative end-joining, and HR, respectively. The KO plants were irradiated with γ-rays, and their radioresistance was evaluated. Although wild-type (WT), lig4, and polq plants showed comparable radioresistance, that of rad51b plants was drastically reduced. The radioresistance of rad51b polq plants was further reduced, whereas that of rad51b lig4 plants was similar to that of rad51b. Under γ-irradiation conditions at which the dry weight of the plants was reduced to 50 %, single base substitutions were predominantly induced in WT, lig4, and polq plants. In contrast, drastic sequence alterations, such as large deletions with or without insertions, chromosome inversions, or translocations, were induced in rad51b and rad51b polq plants. These results suggest that P. patens primarily uses the HR pathway for DSB repair, even in the presence of other pathways. Flow cytometry analysis of the WT and KO plants revealed that the majority of cells subjected to irradiation were in late S/G2 phase, suggesting that the sister chromatid served as a template for HR.