Genetic variations are a crucial source of germplasm heterogeneity, as they contribute to the development of new traits for plant breeding by offering an allele resource. Gamma rays have been widely used as a physical agent to produce mutations in plants, and their mutagenic effect has attracted much attention. Nonetheless, few studies have examined the whole mutation spectrum in large-scale phenotypic evaluations. To comprehensively investigate the mutagenic effects of gamma irradiation on lentils, biological consequences on the M1generation and substantial phenotypic screening on the M2generation were undertaken. Additionally, the study followed the selected mutants into the M3generation to evaluate the agronomic traits of interest for crop improvement. Seeds of lentil varietyMoitreewere irradiated with a range of acute gamma irradiation doses (0, 100, 150, 200, 250, 300, and 350 Gy) to induce unique genetic variability. This research focused on determining the GR50value while considering seedling parameters and examining the status of pollen fertility while comparing the effects of the gamma irradiation dosages. The GR50value was determined to be 217.2 Gy using the seedling parameters. Pollens from untreated seed-grown plants were approximately 85% fertile, but those treated with the maximum dosage (350 Gy) were approximately 28% fertile. Numerous chlorophyll and morphological mutants were produced in the M2generation, with the 300 Gy -treated seeds being the most abundant, followed by the 250 Gy -treated seeds. This demonstrated that an appropriate dosage of gamma rays was advantageous when seeking to generate elite germplasm resources for one or multiple traits. Selected mutants in the M3generation showed improved agronomic traits, including plant height, root length, number of pods per plant, and yield per plant. These investigations will contribute to a comprehensive understanding of the mutagenic effects and actions of gamma rays, providing a basis for the selection and design of suitable mutagens. This will facilitate the development of more controlled mutagenesis protocols for plant breeding and help guide future research directions for crop improvement using radiation-induced mutation breeding techniques.