<p>Mulberry is a critical component of sericulture, as the quality of its leaves directly impacts silkworm growth, cocoon yield, and the quality of silk produced. The application of mutagenesis to induce variability has demonstrated effectiveness in the development of superior mulberry varieties. Increasing genetic diversity within mulberry is crucial for the creation of nutritionally enhanced strains. Gamma irradiation has emerged as a potent mutagenic technique capable of inducing novel traits and promoting genetic permutations and combinations in mulberry, particularly since these traits are polygenic in nature. In this study, S₅₄ mulberry juvenile twigs were subjected to varying doses of gamma irradiation (ranging from 1 kR to 10 kR) to establish putative mutant lines. The resulting putative mutants were cultivated using a randomized block design to generate the M₂ generations. Phytochemical profiling and bioassay analysis were performed to assess the impacts of irradiation. Notably, leaves from cuttings treated with 7 kR exhibited significant improvements in various biochemical parameters, including total proteins, soluble sugars, phenolic compounds, total chlorophyll content, and moisture content across tender, medium, and coarse putative mutant leaves. To evaluate the effectiveness of the putative mutant leaves, a bioassay was conducted utilizing crossbred (PMxNB₄D₂) silkworms. The silkworms that were fed leaves from cuttings treated at 7 kR displayed marked enhancements in commercial traits, such as cocoon weight, shell weight, pupal weight, filament length, denier, and renditta. These findings underscore the potential of the S₅₄ putative mutant in advancing silk production.</p>