Tropical streams on high oceanic islands are characterized by populations of amphidromous gobioid fishes. Adult fish live and breed in freshwater and many spawn at high (>300 m) elevations. Newly hatched fry are swept down to the sea where they develop for a period of time before recruiting to a stream. In analogous habitats in other geographic areas, amphidromous populations have further evolved into landlocked forms (i.e., populations that spend their entire life cycle in freshwater). We analyzed depositional patterns of trace elements in the otoliths of adultLentipes concolor, an amphidromous goby endemic to Hawaii, to determine whether landlocked forms occurred. Otoliths were obtained from fish collected from the Hawaiian stream habitats most likely to harbor landlocked populations—upper elevations of interrupted streams and sections of streams above high waterfalls. A transition from a marine phase to freshwater existence was demonstrated as a decrease from high to low strontium : calcium ratios in the otolith with increasing distance from the otolith's core. In every case, otoliths from these gobies showed evidence of a marine planktonic larval stage, suggesting that even in habitats physically isolated from the sea, these freshwater fish require a marine larval phase.