Landlocking is a process whereby a population of normally diadromous fish becomes limited to freshwater, potentially leading to behavioural, morphological, and genetic changes, and occasionally speciation. The study of recently landlocked populations can shed light on how populations adapt to environmental change, and how such life-history shifts affect population-genetic structure. Kōaro (Galaxias brevipinnis) is a facultatively diadromous Southern Hemisphere galaxiid fish that frequently becomes landlocked in inland lakes. This study compares seven landlocked kōaro populations to diadromous populations from main and offshore islands of New Zealand. Genotyping-by-sequencing was used to obtain genotypes at 18,813 single nucleotide polymorphism sites for each population. Analyses of population structure revealed that most landlocked populations were genetically highly distinct from one another, as well as from diadromous populations. A few particularly isolated island and lake populations were particularly strongly genetically differentiated. Landscape characteristics were measured to test whether lake elevation, size, or distance from the sea predicted genetic diversity or differentiation from diadromous kōaro. While there were no significant relationships indicating isolation-by-distance or isolation-by-environment, we detected a trend toward lower genetic diversity in lakes at higher elevations. Our findings illustrate the critical role that landlocking can play in the structure of intraspecific genetic diversity within and between populations.