Abstract: Endogenous auxin, or indole-3-acetic acid (IAA), plays a central role during root induction in woody plants. The relationship between auxin and root formation and the changes in levels of endogenous hormones in the rhizogenesis of poplar has been studied for many years, but the tissue-specific localization of auxin in rhizogenesis has not been reported. We investigated the distribution of endogenous IAA in shoots of poplar in vitro using immunocytochemistry during root induction. Poplar 741 shoots were rooted at day 10 of in vitro culture on 1/2 MS medium without exogenous growth regulators. The rooting rate was up to 100% at day 14. Before root induction, a weak signal was detected throughout the basal region of the freshly excised shoots. At 8 days after induction, a strong IAA immune reaction was observed in the vascular bundle tissues of the basal region of the shoots, and the vascular bundle tissues of the middle region also showed an IAA signal mainly distributed in phloem and vascular cambium. At 10 days after induction, the cells of the vascular cambium dedifferentiated and developed into visible root primordia, which bore a strong IAA signal. At 12 days after induction, the root primordia developed into adventitious roots and broke through the stem epidermis, with marked IAA signal detected in root tips and root vascular cylinder. We discuss the characteristics of tissue-specific distribution of IAA and the likely transport route during the rhizogenesis of poplar 741 shoots.