To explore and explain the changes of runoff process in the middle and lower Yangtze River mainstream and the two lakes of Dongting and Poyang before and after the operation of the Three Gorges Reservoir (TGR), the runoff data of five hydrological stations, which are Yichang, Jianli, Datong, Qilishan, and Hukou, were used. The stations of Yichang, Jianli, and Datong locate along the mainstream of the Yangtze River, while the other two stations locate at the exit of the Dongting and Poyang Lake, respectively. The runoff changes at each station before and after the TGR's operation and its causes were analyzed. Conclusions include: (1) The annual runoff at each station decreased, except at Qilishan Station, the reduction ratio of runoff at other stations was less than 10% and their changes were not significant; (2) The maximum decrease in monthly runoff at each mainstream station occurred in October, except that at Qilishan Station and Hukou Station it occurred in July and April, respectively; (3) The maximum increase in monthly runoff at each mainstream station occurred in March, while at Qilishan Station and Hukou Station it occurred in January and June, respectively; (4) At Yichang Station, the increase of monthly runoff from January to April resulted from both the inflow increment of the TGR and its operation. The main reason for the decrease in monthly runoff from June to August was the decrease in the inflow of the TGR. The main reason for the runoff change in May, September and October was the operation of the TGR; (5) At Jianli Station, the change of runoff was highly consistent with that at Yichang Station, but the increase of runoff at Jianli Station in each month in winter was greater than that at Yichang Station; (6) At Datong Station, from April to June the change direction of monthly runoff was the same as that at Hukou Station, and the change direction of other months was the same as that at Yichang Station. (7) At Qilishan Station, the main reason for the runoff reductions in July was the reduction of rainfall in Lake Dongting basin, and while the reduction of the diversion from Jingjiang River is the main cause to the runoff reduction in September and October, the reduction of rainfall in Lake Dongting basin also played an important role; (8) At Hukou Station, the decreases in runoff in April and May resulted from the reduction of rainfall in Lake Poyang basin, while the main reason for the runoff decreases in September and October was the reduction of water level in Lake Poyang during the previous months, which led to the early low water level season and the reduction of water storage.