The use of freshwater habitats was examined in three amphidromous goby species of the genus Sicyopterus using otolith microchemistry. Two species were endemic to either New Caledonia or Vanuatu whilst the other was widely distributed. Depositional patterns of strontium (Sr) and barium (Ba) in the otolith of adults were analysed with femtosecond laser ablation inductively coupled plasma mass spectrometry (ICP-MS). The Sr:Ca and Ba:Ca results uncovered three different adult behaviours within the freshwater habitat. Some fishes stayed in elevated locations (square profile); others undertook back-and-forth migrations between higher and lower reaches (up-and-down profile), and finally, others stayed in the lower reaches (constant profile). The consequences of these movements to larval survival or competition for food and territory are discussed. This work brings new knowledge on amphidromous behaviour, and it highlights the necessity of multi-elemental analysis to study amphidromy in freshwater systems.