Climate change aggravates drought and flood disasters, which severely constrains regional water resources utilization and economic development. The Dongting Lake Basin is an important ecological functional area of the Yangtze River Basin, and the study of runoff variation characteristics in this basin can provide a scientific basis for the protection, development, utilization and planning of water resources, as well as coordinated and sustainable development of the region. Based on Mann-Kendall trend test and wavelet analysis method, this study analyzed the multi-time scale variation characteristics of runoff in the control hydrological stations in the Xiangjiang River, Zijiang River, Yuanjiang River and Lishui River in the Dongting Lake Basin during 1960—2022. The results showed that the measured runoff for all four hydrological stations showed an insignificant variation trend, among which Shimen Station in the Lishui River showed a decreasing trend, with a linear regression rate of -0.24 m3/s·a-1; whereas Taoyuan Station in the Yuan River, Taojiang Station in the Zijiang River and Xiangtan Station in the Xiangjiang River all showed an increasing trend, with linear regression rates of 2.42 m3/s·a-1, 0.43 m3/s·a-1 and 3.33 m3/s·a-1, respectively. In terms of temporal distribution, the runoff in these four stations was quite unevenly distributed within the year, with the maximum runoff mainly occurring in June and July and the runoff from April to July accounting for about 50% of the annual runoff, while the runoff in the dry season was relatively low. Moreover, the observed periodicity of runoff in the Lishui River Basin was significantly different from that in the other three river basins, the principal cycle of runoff at the Shimen Station was four years, while that of the other three stations was about 24 years. Notably, the measured runoff in the four sub-basins of the Dongting Lake was synchronized, and the correlation coefficients on annual, seasonal and monthly scales were all above 0.65. In addition, the relationship between precipitation and runoff at different periods remained basically unchanged, indicating that human activities had little influence on the runoff of rivers.