Compared to wide ranges of genetic variation of natural populations, very limited Miscanthus cultivar has been released. This study was the first report on the development of Miscanthus cultivar by means of radiation breeding. Seeds of M. sinensis were initially exposed to gamma rays of 250 Gy for 24 h, generated from a 60Co gamma-irradiator. The irradiated seeds were sown and then the highly tiller-producing mutants were selected for this study. Biomass-related parameters including tiller number, plant height, stem diameter, and leaf number were measured. Ploidy level and internal transcribed spacer (ITS) were investigated to characterize the mutants compared to wild type (WT) Miscanthus. Plant height and tiller number were negatively related, where multi-tillering mutants were relatively short after 4 month growth. However stem diameter and leaf number were greater in mutants. All the materials used in this study were diploid, implying that the mutants with greater tiller numbers and stem diameter were not likely related to polyploidization. Based on the sequence of ITS regions, the mutants demonstrated base changes from the gamma irradiation where G+C content (%) was decreased in the ITS1, but increased in ITS2 when compared to WT sequence. ITS2 region was more variable than in ITS1 in the mutants, which collectively allows identification of the mutants from WT. Those mutants having enhanced tillers and allelic variations might be used as breeding materials for enhanced biomass-producing Miscanthus cultivars.