Background Many postglacial lakes contain fish species with distinct ecomorphs. Similar evolutionary scenarios might be acting on evolutionarily young fish communities in lakes of remote islands. One process that drives diversification in island freshwater fish species is the colonization of depauperate freshwater environments by diadromous (migratory) taxa, which secondarily lose their migratory behaviour. The loss of migration limits dispersal and gene flow between distant populations, and, therefore, is expected to facilitate local morphological and genetic differentiation. To date, most studies have focused on interspecific relationships among migratory species and their non-migratory sister taxa. We hypothesize that the loss of migration facilitates intraspecific morphological, behavioural, and genetic differentiation between migratory and non-migratory populations of facultatively diadromous taxa, and, hence, incipient speciation of island freshwater fish species. Results Microchemical analyses of otolith isotopes (88Sr, 137Ba and 43Ca) differentiated migratory and non-migratory stocks of the New Zealand endemic Gobiomorphus cotidianus McDowall (Eleotridae). Samples were taken from two rivers, one lake and two geographically-separated outgroup locations. Meristic analyses of oculoscapular lateral line canals documented a gradual reduction of these structures in the non-migratory populations. Amplified fragment length polymorphism (AFLP) fingerprints revealed considerable genetic isolation between migratory and non-migratory populations. Temporal differences in reproductive timing (migratory = winter spawners, non-migratory = summer spawners; as inferred from gonadosomatic indices) provide a prezygotic reproductive isolation mechanism between the two ecotypes. Conclusion This study provides a holistic look at the role of diadromy in incipient speciation of island freshwater fish species. All four analytical approaches (otolith microchemistry, morphology, spawning timing, population genetics) yield congruent results, and provide clear and independent evidence for the existence of distinct migratory and non-migratory ecotypes within a river in a geographically confined range. The morphological changes within the non-migratory populations parallel interspecific patterns observed in all non-migratory New Zealand endemic Gobiomorphus species and other derived gobiid taxa, a pattern suggesting parallel evolution. This study indicates, for the first time, that distinct ecotypes of island freshwater fish species may be formed as a consequence of loss of migration and subsequent diversification. Therefore, if reproductive isolation persists, these processes may provide a mechanism to facilitate speciation.