Waterlogging stress seriously constrains crop growth and yield formation. Most dryland crops adapt to waterlogging by forming more aerenchyma in roots to maintain oxygen supply under low oxygen conditions. However, the mechanisms by which waterlogging priming affects aerenchyma formation are still not fully understood. In this study, during the four-leaf stage of wheat, plants were challenged with waterlogging for seven days after 2day priming and 10-day recovery. The secondary roots of wheat were further divided into old nodal roots and newly elongated nodal roots. Under waterlogging stress, for the priming-sensitive cultivar, aerenchyma formation rate in old nodal roots was relatively faster and more sensitive to priming than that in newly elongated nodal roots. Waterlogging priming had a positive effect for the priming-sensitive cultivar on the aerenchyma formation in wheat secondary roots by affecting the generation of signalling substances. Under waterlogging conditions, the aerenchyma formation in old nodal roots of wheat was more correlated with reactive oxygen species (ROS) and ethylene, while in newly elongated nodal roots, it was more correlated with ROS and Ca2 + . Our findings reveal new insights into the physiological mechanisms of waterlogging priming to improve the survival ability of wheat roots under waterlogging environments, and provide biological information on wheat root adaptation to waterlogging.