Tonle Sap Lake is the largest freshwater lake in Southeast Asia. Water development infrastructures are increasingly being constructed in the Lancang-Mekong River Basin, which is a major concern considering its potential impact on Tonle Sap Lake. This study aimed to investigate variations in the area of the lake and discuss their possible linkage to runoff alterations in the Lancang River (Upper Mekong) by comparing runoff at the Yunjinghong hydrological station before and after significant changes in runoff trends that occurred in 2008. First, four commonly used water body extraction methods (MNDWI, NDWI, NDVI, and EVI) were compared and MNDWI was found to provide a better and more stable performance. Based on MOD09A1 data, MNDWI was used to extract the water area of the lake from 2000 to 2014, and characteristics of variations in the area before and after 2008 were analyzed. The water area of Tonle Sap Lake displayed an overall decreasing trend, and specifically decreased by 8.3% during the flood season and by 1.5% on average during the dry season after 2008. Seasonal variations in the water area of Tonle Sap Lake were dominantly influenced by runoff from the Mekong River. Compared with the period 2000-2007, runoff at Yunjinghong station were increased during the dry season (20.74%) and decreased during the flood season (34.25%) between 2008 and 2014. Changes in upstream runoff contributed to runoff at the Stung Treng station in the lower Mekong River by 6.17% (dry season) and −2.41% (flood season). Evidently, the operation of dams in the Lancang River does not primarily account for the area decrease of Tonle Sap Lake during the flood season. In contrast, runoff increase during the dry season mitigates the area decrease of Tonle Sap Lake to a certain extent.