<p>Induction of gynogenesis through un-pollinated ovary culture is an important tool for the production of haploids and doubled haploids in horticultural crops. However, very limited research has been undertaken on the high-throughput induction of haploids and doubled haploids in <i>Chrysanthemum morifolium.</i> Therefore, the current study was carried out to develop an efficient protocol for gynogenesis by optimizing various factors vital to haploid induction. Two cultivars of chrysanthemum, ‘Pusa Centenary’ and ‘Star White’, were chosen. Considering the flower bud developmental stages, half-opened flower buds were more responsive in the induction of gynogenesis than the fully opened and un-opened buds. The induction and development of gynogenic haploid regenerants were significantly increased when ovaries were inoculated on MMS medium (MS + 5% coconut water) rather than the other commercially available media. Among various combinations of growth regulators, ovaries cultured in media containing 1.0 mg L^− 1 TDZ (thidiazuron) along with 0.2 mg L^− 1 NAA (45&#xa0;g L^− 1 sucrose, 0.3&#xa0;g L^− 1 casein hydrolysate, 0.2 mg L^− 1 GA₃, and 8&#xa0;g L^− 1 agar), showed a notably high gynogenic response. Among the various stress treatments, two days of cold pre-treatment at 4&#xa0;°C along with 30 days of dark incubation proved to be highly beneficial for the induction of gynogenesis. By counting the number of chloroplasts in stomatal guard cells, we characterized 15.38% of plants as tri-haploids and 30.76% as hexaploids, whereas 53.84% were mixoploids. These results were reconfirmed by flow cytometry analysis. This study provided new ways to improve the efficiency of haploid induction through unfertilized ovary culture and paved the way for doubled haploid-based breeding in chrysanthemum.</p> Graphical Abstract <p></p>