The Mekong River is one of the largest rivers in the world and hosts the second greatest fish diversity in the world after the Amazon. However, despite the importance of this diversity and its associated biomass for human food security and the economy, different anthropogenic pressures threaten the sustainability of the Mekong River and fish diversity, including the intense damming of the main river. Both the increase in salt-water penetration into the Mekong Delta and the disrupted connectivity of the river may have serious impacts on the numerous freshwater and migratory species. To evaluate the potential of an eDNA approach for monitoring fish diversity, water was sampled at 15 sites along the salinity gradient in the Mekong Delta and along 1500 km of the main stream, from Vietnam to Thailand and Laos. A total of 287 OTUs were recovered, of which 158 were identified to the species level using both reference sequences available in GenBank and references obtained locally. Agglomerative hierarchical clustering and PCA identified up to three main species assemblages in our samples. If the transition from brackish to freshwater conditions represents the main barrier between two of these assemblages, more surprisingly, the two other assemblages were observed in the freshwater Mekong, with a spatial disjunction that did not match any biogeographic ecoregion or the Khone falls, the latter thought to be an important fish dispersion barrier. Between 60% and 95% of the freshwater species were potamodromous. This pioneer eDNA study in the Mekong River at this geographical and ecological scale clearly confirmed the potential of this approach for ecological and diversity monitoring. It also demonstrated the need to rapidly build an exhaustive Mekong fish barcode library to enable more accurate species’ assignment. More eDNA surveys can now be expected to better describe the ecological niche of different species, which is crucial for any models aimed at predicting the impact of future damming of the Mekong.