The exposure of cancer cells to ionizing radiation results in potentially lethal DNA lesions. For this reason, identification and quantification of various lesions have intensively been investigated. It has also been anticipated that DNA lesions may affect not only the chemical but also the mechanical integrity of the double helix. However, the relationship between DNA damage and mechanics has not been studied. Here, the mechanical properties of DNA damaged by ionizing radiation are examined at a single-molecule level by stretching lambda-phage DNA molecules that have been exposed to gamma-radiation. A simple-stretching method using Atomic Force Microscopy (AFM) not only identifies the types of DNA lesions but also provides information about the mechanical instability of damaged DNA against intact DNA. The results include the elastic properties of damaged DNA with single strand breaks (SSBs), double strand breaks (DSBs), and multiple-lesion clusters. The elasticity of irradiated DNA is changed compared to that of intact DNA. Specifically, consecutive stretching cycles of DNA containing multiple SSBs progressively shorten the width of the overstretching B-S transition. This originates from force-induced melting off of single-stranded DNA fragments, which upon consecutive stretching cycles converts the double helix into a hybrid structure with a growing number of single stranded gaps. Closely spaced SSBs on opposite strands, upon stretching, result in a rupture of the double helix at a decreased force of approximately 200 pN and other clustered lesions result in lowering the force at which force-induced melting of the double helix occurs. Taken together, our results suggest that single-molecule force spectroscopy may become a useful nanoscale DNA diagnostic tool.