In bread wheat (Triticum aestivum L.), gamma-ray mutants have been used to obtain novel agronomic traits and breeding materials. The design of markers linked to mutant phenotypes is an essential step in proceeding with maker-assisted selection. This study aimed to develop an efficient method to identify structural variations responsible for mutant phenotypes of gamma-irradiated mutants. We irradiated the Japanese bread wheat variety “Kitahonami” with gamma-rays, and then obtained a grain-hardness mutant and a pre-harvest sprouting tolerance mutant. Gamma-ray often causes structural variants such as deletions. To identify genomic regions that caused the mutant phenotypes, we applied whole-genome sequencing to these two mutants and wild-type to detected structural variants by comparing the depth-of-coverage between wild-type and mutants. In the grain hardness mutant, a 130 Mbp deletion was detected on the short arm of chromosome 5D, where known grain hardness genes Pina and Pinb are located. We also identified a 67.8 Mbp deletion on chromosome 3B in the pre-harvest sprouting mutant. This region contained known pre-harvest sprouting related gene Viviparous-B1 and potential gibberellic acid signaling gene GRAS family transcription factor. The segregation analysis with the molecular markers showed that the mutant phenotypes were associated with these deletions. In addition, our simulation study found that our method can effectively detect deletions longer than 100 kbp, and 2.5× depth-of-coverage is enough to detect large deletions. Our approach based on the depth-of-coverage would promote the usage of gamma-irradiated mutants in breeding with marker-assisted selection.