Spatial and temporal variation of nine elements (Ba, Ca, Cu, Mg, Mn, Na, Se, Sr, and Zn) and some additional environmental parameters (salinity, water levels, and conductivity) were examined over two 2,000 km along the length of the main Mekong River channel from Luang Prabang in Northern Lao PDR to the Mekong Delta in Vietnam. Longitudinal profiles of some dissolved elemental concentrations varied significantly along the river channel. For example, Sr concentration in marine water was up to 83 times higher than in fresh water. By contrast, Ba concentration was around 4 times higher in fresh water than in marine water, but was significantly influenced by inflows from major tributaries in Cambodia (dilution effects). We found dissolved Ba was elevated in low salinity. Furthermore, Sr concentration was more variable than Ba, especially in the Mekong Delta due to different tidal influences in each of the Delta branches. Selenium was only present along the coast and one river branch in the delta. The variation in water chemistry of the Mekong main channel identified through this study provides information that can be used in the interpretation of regional fish movements using otolith chemistry, including across the freshwater, estuarine and marine interface. Accurate interpretation is essential given the Lower Mekong Basin has a high biodiversity that is under threat from rapid development, and substantial knowledge gaps exist on the life history requirements of numerous fish species.