The most direct approach in breeding triploid Populus is crossing allotetraploids with diploids. However, the lack of allotetraploid Populus restricts application of this approach. In this investigation, zygotic chromosome doubling was induced with colchicine and high temperatures to produce ((Populus pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis)) allotetraploids. We screened 6 and 25 tetraploid individual offspring from the colchicine and high-temperature treatments respectively, indicating that both colchicine and high temperature are effective for tetraploid production by zygotic chromosome doubling of Populus. Developmental characteristics of seed hairs in the ovaries were temporally associated with zygotic development, which was used to successfully guide the colchicine and high-temperature treatments. During certain stages of hair development, the efficiency of tetraploid production was significantly high. However, efficiency of production was not significantly influenced by other factors, i.e. colchicine concentration, temperature or duration of high-temperature treatment. Size and frequency of leaf stomata between tetraploid and diploid plants were significantly different, suggesting that this character can be altered via genomic increase in material. The allotetraploids produced in this investigation, having different genotypes, provide important parental germplasms for further triploid breeding.