Abstract: Breeding of photoperiod or thermo-sensitive male sterile line is the key for the development of two-line hybrid rice. The allele of tms5 is the major TGMS genetic resource for two-line hybrid rice breeding in China. Targeted editing on TMS5 of fertile rice using CRISPR/Cas-9 technology could effectively create thermo-sensitive genic male sterile rice. This study used the approach to edit the TMS5 gene successfully. The CRISPR/Cas-9 vector of TMS502 was constructed, then the T-DNA was transformed into GH89. Ten transgenic plants were thus regenerated, with 6 of them containing either the insertion or deletion mutations. The T₀ plant of tms5-l was a homozygous mutant, and tms 5-4 a bi-allelic mutant. Both of them displayed thermo-sensitive male sterile phenotype. There were 4 homozygous mutant genotypes in the T₁ generation of the non-transgenic plants. The mutants showed the same characteristics as shown in the T₀ generation without any new mutations. Male fertility of T₂ generation of tms5 mutants was tested in natural high temperature environment and in phytotrons at 3 temperature levels. The results indicated that the critical sterility-inducing temperature of the tms5 sterile lines was approximately 24 C, which satisfied the requirements for two-line male sterile line breeding. The successful conversion of GH89 line into a two-line male sterile rice using the gene editing technology suggested that CRISPR/Cas-9 could be applied to facilitate the development of two line hybrid rice breeding in the future.