The Lower Mekong Basin has had extensive hydropower dam development, which changes its hydrologic conditions and threatens the exceptional aquatic biodiversity. This study quantifies the degree of hydrologic change between pre-impact (1965–1968) and post-impact (2018–2021) peak hydropower development in two major tributaries of the Lower Mekong Basin—the Sekong River, with the fewest dams, and the Sesan River, with the most dams. Both rivers have historically supported migratory fishes. We used daily pre- and post-impact data and the Indicators of Hydrologic Alteration framework to evaluate streamflow changes from dam development. We found significant changes in low- and high-magnitude flows in the pre- and post-impact periods of dam development. For the Sekong River, minimum flow had large fluctuations, with increases of 290% to 412% compared to the pre-impact period, while the Sesan River’s minimum flow ranged from 120% to 160% more than pre-impact. Dry season flows increased by 200 ± 63% on average in the Sekong River, which was caused by releases from upstream dams. Meanwhile, the Sesan River’s dry season flows increased by 100 ± 55% on average. This study indicates that seasonal flow changes and extreme flow events occurred more frequently in the two basins following dam construction, which may threaten the ecosystem’s function.