The availability of diversified germplasm resources is most important for developing improved rice varieties with higher seed yield and other desired agronomic traits. In this study, the effect of subculture time, 2,4-Dichlorophenoxyacetic acid (2,4-D) concentration and variant explants on the frequencies of plant regeneration and mutagenesis were investigated, and a new somatic mutagenesis breeding method for rice with the immature inflorescences as explants, 3 cycles of subculture time (about 75 d) and 4.0 mg/L 2,4-D as the preferred mutagenic concentration was developed. Then, the parents of hybrid rice, Zhu1S and Zhong 9B, were mutagenized in this new method, and 2 elite semi-dwarf somatic lines, SV14S and SV9B, were successfully isolated, which will provide high-quality germplasm resources for follow-up cross-breeding. The breeding achievements made in this study further confirm that this rice somatic mutagenesis breeding method is a simple and effective breeding method.