The application of male sterility has facilitated the commercialization of heterosis in rice, resulting in an enhanced yield and output of this staple food crop. The application of three-line hybrid rice technology is decreasing due to limited heterosis, lack of good combiner in cytoplasmic male sterile lines, poor reproducibility of hybrid seeds and limited commercial acceptance. Two-line heterosis breeding can overcome these issues. Two-line breeding allows for more diverse germplasm as parents, including any line as female and 97% of germplasm as male. Moreover, two-line breeding can lead to lower production costs. Systematic research can enable the widespread deployment of two-line hybrid rice technology. Identifying the novel genetic, molecular, and cellular factors and understanding the regulatory networks regulating male sterility in rice is vital for maximizing heterosis and ensuring global food security. In this review, we have briefly discussed the types of environment-sensitive genetic male sterility systems for two-line hybrid seed production. Furthermore, we have discussed the genetic, molecular and cellular basis for the thermosensitive genetic male sterility (TGMS) mechanism. We have provided insights on molecular and biotechnological interventions such as CRISPR and omics techniques in the TGMS system to address the existing concerns and to overcome the problems related to two-line rice breeding.