Effects of the native GnRHs and various agonists have been evaluated on the spawning of an Indian catfish, Heteropneustes fossilis. This study tested salmon (s) GnRH agonists and mammalian (m) GnRH agonists where a D-amino acid residue was substituted alone at position 6 or the C-terminal was modified with ethylamide. GnRH agonists with a combination of these structural modifications were also evaluated separately for their effect on the spawning of the catfish. Native sGnRH, [Pro9 NEt]-sGnRH agonist and chicken (c) GnRH-II exhibited similar activity and induced spawning within 14–18 h at a dose of 100 μg kg−1 body weight (BW). [D-Lys6]-sGnRH agonist and [D-Lys6 Pro9 NEt]-sGnRH agonist, induced spawning at a dose of 100 μg kg−1 BW and 1 μg kg−1 BW, respectively. The most notable observation in this study was the ineffectiveness of [D-Ala6]-mGnRH agonist and [Des Gly10 D-Ala6 Pro9 NEt]-mGnRH agonist. The results obtained suggest that substitution at position 6 alone, and in conjunction with an ethylamide-based modification at the C-terminal in the native sGnRH structure, increases the potency of the tested agonists to induce spawning in the catfish. This study also discusses the potential use and incorporation of cGnRH-II for the development of more generic spawning induction therapies.