Xiangxi River is a typical tributary of Three Gorges Reservoir (TGR) in China. Based on field observations in 2010, thermal stratification was significant in most months of the year. Through field data analysis and numerical simulations, the seasonal and spatial variation of thermal stratification as related to the impact of the operation of TGR were investigated. Thermal stratification was most pronounced from April to September in the Xiangxi River tributary. Air temperature (AT) and water level (WL) were the two dominant variables impacting thermal stratification. AT affected the surface water temperature promoting the formation of thermal stratification, and high WLs in TGR deepened the thermocline depth and thermocline bottom depth. These results provide a preliminary description of the seasonal variation and spatial distribution of thermal stratification, which is important for better understanding how thermal stratification affects algae blooms in Xiangxi River.