<p>The three-line system of hybrid rice has been a widely adopted method used to develop high-yielding hybrid varieties. The system encompasses a cytoplasmic male sterile line (A line), a maintainer to the male sterile line (B line), and a fertility restorer line (R line). For the first time, the study validates a comparative assessment of an efficient anther culture technique utilizing F₁ hybrids derived from crosses between (A × R) and (B × R) genotypes. In the above hybrid system, A × R leads to heterosis resulting in hybrid rice production while B × R crosses envisioned to develop superior maintainer or restorer lines. The ANOVA along with Duncan’s mean supported N6 medium fortified with 2.0&#xa0;mg L⁻¹ 2,4-di-chlorophenoxyacetic acid and 0.5&#xa0;mg L⁻¹ 6-benzylaminopurine resulted in maximum callusing on the 7th d with a callusing frequency of 47.63% for A × R (~ 475 anthers responded out of 1000 used) and 34.4% (~ 340 anthers responded out of 1000 used) for B × R. Similarly, MS medium supplemented with 0.5&#xa0;mg L⁻¹ 1-naphthalene acetic acid, 2.0&#xa0;mg L⁻¹ 6-benzylaminopurine, and 1.0&#xa0;mg L⁻¹ kinetin was identified to be most effective and optimal for shoot regeneration. The cross—A × R—showed the highest green shoot regeneration frequency (80.34%) while the addition of proline (5.0&#xa0;mg L⁻¹) to MS₄ media in the B × R cross doubled its green regeneration frequency (35.5 to 70.1%). Surprisingly, no haploids were produced from B × R derivatives while it was 15 in the case of A × R. Additionally, it examines the influence of cytoplasmic sterility factors on androgenesis. Moreover, it can be focused on developing new elite DH lines that can help generate new diversified parental lines in hybrid rice production.</p> Graphical Abstract <p></p>