AbstractReynoutria japonica (Japanese knotweed) is a highly invasive female plant that primarily reproduces through vegetative regeneration during secondary distribution. Despite producing a substantial number of viable hybrid seeds, the occurrence of seedlings and young plants in the wild remains rare; the reasons for this rarity are unclear. Environmental intolerance and chromosomal anomalies during hybridisation can impede seedling development. This study evaluates the combined impact of these factors by examining substrate, temperature, and light effects on germination, as well as assessing seedling genome sizes and viability. Seeds were cultivated in both natural and controlled environments to manage external influences. Flow cytometry was employed to evaluate chromosomal arrangements. The final germination was notably high at 99%, and 68% of seedlings thrived under controlled conditions, emphasising hybrid seedling viability regardless of highly polyploid levels ranging from pentaploid to aneuploid (2C DNA genome size from 5.17 to 11.95 pg). Thus, it is evident that seeds can germinate and produce vital seedlings despite various chromosomal sets. Even fluctuating temperatures and type of substrate do not limit seed germination. However, these results were obtained under laboratory conditions, with seeds and seedlings receiving regular irrigation. A significant seedling mortality rate (99%) was observed in the field experiment, and the final germination was also low there (15%). Observations suggest that water stress might be the cause of this mortality. Further research on water stress is necessary because it could be the primary factor limiting the successful generative spread of the knotweeds.