Processes responsible for population structuring across spatial and temporal scales represent key components in understanding speciation and evolution. We use a hierarchical approach to investigate the degree and mechanisms of structuring in landlocked and diadromous populations of the facultatively amphidromous fish Galaxias brevipinnis across various temporal and spatial scales in southern New Zealand. To determine long-term structuring, multiple lakes and coastal sites were compared genetically. Short-term structuring was assessed using otolith microchemistry for a subset of sites, and behavioural mechanisms driving population structuring were assessed via larval distributions. Genetic data show that lakes foster divergence of lake-developing populations from each other and from coastal stream populations, whereas there is relatively little structuring within coast or lake populations. However, otolith analyses indicate that on a shorter time scale, most larvae do not disperse, i.e. recruitment is local. Thus, lake and coastal populations show a distinct meta-population structure based on catchment, in contrast to the prevailing assumption of widespread dispersal, with implications for management. Most larvae were distributed in river plumes, suggesting that a simple larval behavioural mechanism, e.g. positive rheotaxis, may result in larval retention within catchments and lakes. However, not all larvae were retained in plumes, creating opportunities for genetic exchange within-lake or among coastal sites. Genetic divergence of lake populations as a consequence of landscape and behaviour provides an insight into the potential of G. brevipinnis to diversify and speciate, when landscape and circumstances align, and also has implications for the management of this and other facultatively amphidromous species.