A crucial technique in crop improvement is mutagenesis, which creates genetic variation to produce new plant varieties with enhanced nutritional value, disease resistance, and stress tolerance. By changing the structure of DNA, induced random mutagenesis—achieved by physical (gamma rays, X-rays, UV radiation) and chemical (EMS, MMS, sodium azide) mutagens—speeds up reproduction. Directly exposing seeds, pollen, or entire plants to mutagens is known as “in vivo mutagenesis.” Desired traits are then assessed in the field. Using plant tissue culture, in vitro mutagenesis is typically carried out at the cellular level. It offers early and effective variant selection as well as more control over the induction of mutations. Crop improvement in cereals (rice, wheat), legumes (soybean, lentil), oilseeds (rapeseed), fruits (banana, strawberry), and vegetables (tomato, cassava) has been greatly aided by these techniques, resulting in commercially successful mutant varieties with improved agronomic performance. Mutagenesis has drawbacks despite its advantages, such as the randomness of mutagenic events, the rare chance of getting advantageous features, the possibility of genetic instability, and time-consuming mutant screening. However, advances in molecular markers and genome sequencing has led to improvements in mutation detection and enhanced selection efficiency, resulting in a more successful mutation breeding programs. With a focus on its use in modern crop breeding, this chapter elucidates the significance, methods, uses, and limitations of in vivo and in vitro mutagenesis.