Neo-tetraploid rice, which developed from the progenies of autotetraploid hybrid by our research group, is a useful germplasm with high fertility and strong heterosis when they crossed with other autotetraploid rice lines. The CRISPR/Cas9-mediated TMS5 gene editing system has been widely used in diploid rice, but there are few reports in tetraploid rice. Here, we used CRISPR/Cas9 technology to edit the TMS5 gene, which is a temperature sensitive gene controlling the fertility in diploid rice, in neo-tetraploid rice to develop male sterile lines. Two mutant lines, H2s and H3s, were developed from the gene editing and displayed characteristics of thermo-sensitive genic male sterility. The daily mean temperatures of 23 °C to 26 °C were found to be critical for sterility (restrictive temperature) in H2s and H3s under both controlled (growth chambers) and natural growing conditions (field). Cytological observation showed the anther dysplasia appeared later in H2s and H3s than that of the TMS5 mutant of diploid rice (E285s) under the same conditions. Then these mutant lines, H2s and H3s, were crossed with tetraploid rice to generate F(1) hybrids, which exhibited obvious advantages for effective number of panicles, total grains and seed setting. The high levels of hybrids heterosis were maintained for several generations that can save seed cost. Our research provides an effective way of developing thermo-sensitive genic male sterility (TGMS) lines of tetraploid rice using gene editing, which will accelerate the utilization of polyploid heterosis.