Two-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient. Here, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5–1 and tms5–2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22 °C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26 °C, their pollen was sterile and no grain was set. F1 hybrids derived from the crosses between YK17S (tms5–1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits. The YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.