Here, we explored the mutual regulation of radical oxygen species (ROS) and autophagy in wheat (Triticum aestivum L.) roots under hypoxia stress. We also analyzed differences between the responses of the stele and the cortex in the two wheat cultivars Huamai 8 (waterlogging-tolerant) and Huamai 9 (waterlogging-sensitive) to hypoxia stress. In situ detection and ultracytochemical localization analysis in wheat roots showed that hypoxia stress caused greater increases in ROS levels and the expression levels of alternative oxidase (AOX) and antioxidant enzymes in the stele than in the cortex. The analysis of exogenous ROS addition and the inhibition of its production revealed the pivotal roles played by ROS in autophagy. Moreover, transmission electron microscopy and qRT-PCR analysis indicated that the stele had a higher level of autophagy than the cortex and that the two wheat cultivars primarily differed in the type and number of autophagosomes. Additional research revealed that autophagy could remove excess ROS, as pre-treatment with the autophagy inhibitor 3-methyladenine increased ROS levels in roots and the addition of the autophagy inducer rapamycin reduced root ROS levels. In conclusion, hypoxia stress induced ROS accumulation in wheat roots where ROS acted as an autophagy signal. Furthermore, higher levels of autophagy and antioxidant enzyme expression in the stele facilitated the elimination of oxidative damage caused by excessive ROS and thereby increased cell survival; in the cortex, a large number of cells died and formed aerenchyma.