The incorporation of nanomaterials into agricultural biotechnologies has introduced a new approach to modern agriculture. Among the most widely used nanoparticles in agricultural nanotechnology are zinc oxide (ZnO) nanoparticles (NPs). This study aims to evaluate the effects of ZnO NPs, on donor plant growth, and in vitro gynogenesis efficiency. The plant materials used in this study were long-day onion cultivars, namely cv. ‘Delfos’, cv. ‘Nun 7194’, and the Suluova genotype. Overall, ZnO NPs treatments significantly influenced morphological characteristics in all genotypes. The 60 mg L^-¹ ZnO NPs treatment generally resulted in the highest values for plant growth parameters, while 80 mg L^-¹ was more effective for leaf length and for some flowering traits in cv. ‘Delfos’ and cv. ‘Nun 7194’. In contrast, maximum leaf width in cv. ‘Nun 7194’ was obtained at 100 mg L^-¹. These findings indicate that the optimal ZnO NPs concentration varied among genotypes and traits, with 60 mg L^-¹ ZnO NPs treatment emerging as the more effective. Furthermore, this concentration also exhibited more efficiency in in vitro gynogenesis, of the eight putative gynogenic plantlets exhibiting both root and shoot development, three from cv. ‘Delfos’, three from cv. ‘Nun 7194’, and two from the Suluova genotype.