Barro Colorado Island is located in the man-made Gatun Lake in the middle of the Panama Canal. The island was formed when the waters of the Chagres River were dammed to form the lake in 1913. When the waters rose, they covered a significant part of the existing tropical forest, but certain hilltops remained as islands in the middle of the lake. It has an area of 15.6 km2 (6.0 sq mi).

Gatun Lake is a freshwater artificial lake to the south of Colón, Panama. At approximately 26 m (85 ft) above sea level, it forms a major part of the Panama Canal, carrying ships 33 km (21 mi) of their transit across the Isthmus of Panama. It was created June 27, 1913 when the gates of the spillway at Gatun Dam were closed. The water level was about 14.6 m (48 ft) above sea level and was expected to fill to operating levels in the next six months.