Thermal stratification has a significant impact on water quality and ecological characteristics. Reservoir operation and climate change have an effect on the thermal regime. The Jinpen Reservoir is a large canyon-shaped reservoir located in Shaanxi Province with a strong thermal stratification, which resulted in an anaerobic condition in the hypolimnion. We used a hydrodynamic module based on MIKE 3 to simulate the thermal structure of the Jinpen Reservoir and study the relationship between the thermal regime, reservoir operation and climate change. Based on the daily hydrological and climatic data from 2004 to 2013, we made 13 hypothetical simulated conditions that included extreme change of inflow volume, water level, air temperature, radiation, inflow water temperature and selective withdrawal to explore the effect of different factors on the thermal regime. The results showed that the period of thermal stratification, water column stability and surface water temperature were influenced by these factors. With the increase of air temperature, the simulation results indicated a stronger thermal stratification and a higher surface water temperature, which could cause water safety problems. Deep withdrawal could decrease water column stability and prompt water column mixing early, which could be used by reservoir managers to optimize the reservoir operation.