To improve our knowledge of the migration pathway of a highly threatened fish species along the Mekong River, strontium isotope ratios (87Sr/86Sr) and 18 trace element concentrations were measured in the water and in the otoliths of an anadromous catfish, Pangasius krempfi, to infer its natal origin and potential migration pathways. Water was sampled at 18 locations along the mainstream, tributaries and distributaries of the Mekong River. To check for accuracy and precision, measurements of the 87Sr/86Sr ratios and trace element concentrations were then compared in two laboratories that use different analytical methods. Differences in trace element concentrations between locations were not significant and could not, therefore, be used to discriminate between migration pathways. However, the Mekong mainstream, tributaries and distributaries could all be discriminated using Sr isotopes. The 87Sr/86Sr profiles recorded in P. krempfi otoliths showed that there were three contingents with obligate freshwater hatching and variable spawning sites along the Mekong mainstream, from Phnom Penh (Cambodia) to Nong Khai (Thailand) or further. After hatching, the fish migrated more or less rapidly to the Mekong Delta and then settled for most of their lifetime in brackish water. Spawning habitats and migration routes may be threatened by habitat shifts and the increasing number of hydropower dams along the river, especially the contingents born above Khone Falls (Laos). The conservation of P. krempfi, as well as other migratory fish in the Mekong River, requires agreements, common actions and management by all countries along the Mekong River. This study highlighted the importance of using both Sr/Ca and 87Sr/86Sr ratios to understand life history of anadromous fishes as the 87Sr/86Sr ratio in the water was shown to be less effective than the Sr/Ca ratio in identifying movements between different saline areas.