Abstract: At present, the goal of rice breeding has shifted from one-sided emphasis on yield in the past to comprehensive and balanced development of many traits such as high quality, high yield and multi-resistance. It is an inevitable trend for future breeding to comprehensively utilize the identified favorable genes that control excellent agronomic traits. There are obvious limitations in the traditional technical thinking of cultivating pure-line parents when pyramiding multiple genes controlling favorable traits in rice. In contrast, the development of multi-line varieties can avoid these limitations and is regarded as an important technical strategy to break through the bottleneck of current rice breeding. The application of recessive genic male sterile lines in the backcross breeding of multi-line varieties can greatly improve breeding efficiency and help to break the linkage drag. In this study, the TDR gene was site-specific knocked out in japonica rice using the CRISPR-Cas9 system. Positive transgenic plants were obtained by hygromycin resistance marker screening, and sterile lines free of transgenic components were acquired via sequencing and PCR analysis. A method for the propagation of homozygous male sterile lines, as well as a technical scheme for breeding multi-line varieties using this recessive genic male sterile line were obtained. The generation of novel rice sterile materials by site-specific knockout of the TDR gene using CRISPR-Cas9 technology, together with the matched propagation method for sterile lines and technical scheme established for multi-line variety cultivation, possesses important reference value for the breeding and application of rice multi-line varieties.