The Three Gorges Reservoir (TGR) began to store water in 2003. The water level reached 135 m in 2003, 156 m in 2006, 172 m in 2008 and 175 m (target level) in every year from 2010 through 2013. Impacts of dams on the environment are a concern all over the world. A major concern for reservoirs is phosphorus and possible eutrophication. Therefore, total phosphorus (TP) in the TGR mainstem was determined at five stations (S1–S5) from 1998 through 2013 to investigate variations of phosphorus with water level elevations. Results revealed that a new spatial and seasonal TP distribution has occurred after the impoundment. TP levels in the reach between S3 (nearly 300 km from the dam) and S5 (near the dam) have decreased sharply compared with those before the impoundment. The reduction degree of TP in wet season was greater than in dry season. Additionally, TP spatially decreased from upstream to the dam after the impoundment. The reduction of TP levels was mainly attributed to the settling of suspended solids and adsorbed phosphorus. These findings will inform research about transport and fate of phosphorus in TGR and Yangtze River. The challenge remains to implement measures to limit the release of phosphorus from sediments.