Ratoon rice is a labor-saving and high-efficiency rice cultivation system. Under traditional continuous flooding irrigation, it not only consumes a large amount of water but also suffers from inhibited root vitality due to long-term root hypoxia. As a water-saving irrigation regime, alternate wetting and drying (AWD) has been widely applied in rice production. To explore the response of root growth in ratoon rice to AWD, a conventional indica rice variety Huanghuazhan and a hybrid indica rice variety Liangyou 6326 were used as experimental materials, with two irrigation treatments set up: conventional flooding (CF, control) and alternate wetting and drying (AWD, with a lower limit of soil water potential at -15±5 kPa). A systematic investigation was conducted on the effects of AWD on root vitality, root morphological characteristics and anatomical structure at key growth stages of the main crop season and ratoon season. The results showed that AWD had no significant effect on plant height of both Huanghuazhan and Liangyou 6326, but significantly reduced the maximum tiller number and ineffective tiller ratio in the main crop season. The effect of AWD on root vitality varied with rice varieties. Root vitality of hybrid rice Liangyou 6326 was significantly increased by 82.8% and 103.1% at the peak tillering stage and full heading stage of the main crop season, respectively, whereas no significant change was observed in conventional rice Huanghuazhan. Under AWD, root dry weight showed no significant difference at most growth stages except a significant decrease at maturity of the main crop season, while root morphology was altered, represented by significant reductions in total root length, total root surface area and total root volume. Among roots of different diameter classes, the length, surface area and volume of medium and coarse roots decreased significantly, accompanied by an elevated proportion of fine roots. In the main crop season, AWD significantly decreased the adventitious root diameter by 9.6%~25.3% at full heading stage, and significantly increased the stele diameter and metaxylem area of Huanghuazhan by 10.78% and 14.07%, respectively. In the ratoon season, only the metaxylem area increased significantly at full heading stage, with a greater increment in Liangyou 6326 than in Huanghuazhan. In conclusion, AWD can effectively suppress the occurrence of ineffective tillers in the main crop season, enhance root vitality of hybrid rice, and optimize root morphological and anatomical structure (e.g., increased proportion of fine roots, aerenchyma area and metaxylem area), thereby improving the capacity and efficiency of root water and nutrient uptake. These findings provide a theoretical basis for the implementation of water-saving cultivation practices in ratoon rice production.