Abstract. The rapid rate of water infrastructure development in the Mekong Basin is a cause for concern due to its potential impact on fisheries and downstream natural ecosystems. In this paper, we analyze the historical water levels of the Mekong River and Tonle Sap system by comparing pre- and post-1991 daily observations from six stations along the Mekong mainstream from Chiang Saen (northern Thailand), to Stung Treng (Cambodia), and the Prek Kdam station on the Tonle Sap River. Observed alterations in water level patterns along the Mekong are linked to temporal and spatial trends in water infrastructure development from 1960 to 2010. We argue that variations in historical climatic factors are important, but they are not the main cause of observed changes in key hydrological indicators related to ecosystem productivity. Our analysis shows that the development of mainstream dams in the upper Mekong Basin in the post-1991 period may have resulted in a modest increase of 30-day minimum levels (+17%), but significant increases in fall rates (+42%) and the number of water level fluctuations (+75%) observed in Chiang Saen. This effect diminishes downstream until it becomes negligible at Mukdahan (northeast Thailand), which represents a drainage area of over 50% of the total Mekong Basin. Further downstream at Pakse (southern Laos), alterations to the number of fluctuations and rise rate became strongly significant after 1991. The observed alterations slowly decrease downstream, but modified rise rates, fall rates, and dry season water levels were still quantifiable and significant as far as Prek Kdam. This paper provides the first set of evidence of hydrological alterations in the Mekong beyond the Chinese dam cascade in the upper Mekong. Given the evident alterations at Pakse and downstream, post-1991 changes could also be directly attributed to water infrastructure development in the Chi and Mun basins of Thailand. A reduction of 23 and 11% in the water raising and falling rates respectively at Prek Kdam provides evidence of a diminished Tonle Sap flood pulse in the post-1991 period. Given the observed water level alterations from 1991 to 2010 as a result of water infrastructure development, we can extrapolate that future development in the mainstream and the key transboundary Srepok, Sesan, and Sekong sub-basins will have an even greater effect on the Tonle Sap flood regime, the lower Mekong floodplain, and the delta.