Abstract： Objective We identify ionizing radiation‐induced mitochondrial DNA (mtDNA) deletions in human lymphocytes and their distribution in normal populations. Methods Long‐range polymerase chain reactions (PCR) using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy 60Co γ‐rays. Limited‐condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long‐range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy 60Co γ‐rays, and real‐time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated.Results Two mtDNA deletions, a 7455‐bp deletion (nt475‐nt7929 in heavy strand) and a 9225‐bp deletion (nt7714 ‐nt369 in heavy strand), occurring between two 8‐bp direct repeats, were identified in lymphoblastoid cells using long‐range PCR, limited‐condition PCR and sequencing. These results were also observed for 60Co γ‐rays irradiated human peripheral blood cells. Conclusion Two novel mtDNA deletions, a 7455‐bp deletion and a 9225‐bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors’ age, but was independent of gender.