Abstract: The effects of root cutting length, incision sealing methods, and transplant methods on the rootling establishment and growth of Populus yunnanensis were studied for setting up an efficient and stable rejuvenation technique system for rootling cultivation by taking root segments as propagation materials. The results showed that the root cutting length significantly affected the number of root suckers and the number of adventitious roots, the root suckers and adventitious roots were increased along with the increment of the length of root cuttings, whereas the height and basal diameter of the rootlings were decreased with the length increment of the root cuttings. The incision sealing methods also had significant effect on the height, basal diameter and the growth of root suckers, no sealing on the either end of the cuttings resulted in the best effect. There were no significant effects of the transplant methods on the numbers of rootlings, the adventitious roots, the height and basal diameter of the rootlings, but the effect of oblique burying of the root cuttings was slightly better than that of the horizontal burying on the number of rootlings, the height and basal diameter of the rootlings.