Colchicine is widely used as a mutagen to induce production of diploid gametes in plants. However, whether colchicine affects induced pollen viability remains unclear. To clarify whether colchicine affected the viability of induced pollen, we induced production of diploid pollen by colchicine, followed by pollen germination in vitro and crossing induced pollen with normal gametes to produce triploid in Populus tomentosa Carrière. The results showed that the predominant meiotic stages and the number of colchicine injections had significant effects on the occurrence rates of induced 2n pollen. When the colchicine injection was given at diakinesis, a significant decrease in the pollen production per bud was observed (p < 0.001). The morphology of the colchicine-induced 2n pollen was similar to that of the natural 2n pollen in its ectexine structure. The pollen germination experiments revealed that there was also no significant difference in germination rates between the induced diploid pollen and natural 2n pollen grains, and 68 triploids were created by crossing colchicine-induced pollen. Our findings revealed that colchicine injection could induce P. tomentosa to produce 2n pollen and will not lead to dysfunction of induced diploid pollen.