Background: Lake (sic)ingvallavatn in Iceland, a part of the river Olfusa drainage basin, was presumably populated by brown trout soon after it formed at the end of the last Ice Age. The genetic relatedness of the brown trout in (sic)ingvallavatn to other populations in the Olfusa drainage basin is unknown. After the building of a dam at the outlet of the lake in 1959 brown trout catches declined, though numbers have now increased. The aim of this study was to assess effects of geographic isolation and potential downstream gene flow on the genetic structure and diversity in brown trout sampled in several locations in the western side of the watershed of River Olfusa. We hypothesized that brown trout in Lake (sic)ingvallavatn constituted several local spawning populations connected by occasional gene flow before the damming of the lake. We also estimated the effective population size (NE) of some of these populations and tested for signs of a recent population bottleneck in Lake (sic)ingvallavatn. Methods: We sampled brown trout inhabiting four lakes and 12 rivers within and near the watershed of River Olfusa by means of electro- and net- fishing. After stringent data filtering, 2,597 polymorphic loci obtained from ddRADseq data from 317 individuals were ascertained as putative neutral markers. Results: Overall, the genetic relatedness of brown trout in the Olfusa watershed reflected the connectivity and topography of the waterways. Ancestry proportion analyses and a phylogenetic tree revealed seven distinct clusters, some of which corresponded to small populations with reduced genetic diversity. There was no evidence of downstream gene flow from Lake (sic)ingvallavatn, although gene flow was observed from much smaller mountain populations. Most locations showed low NE values (i.e., similar to 14.6 on average) while the putative anadromous trout from River Sog and the spawning population from River Oxara, that flows into Lake (sic)ingvallavatn, showed notably higher NE values (i.e., 71.2 and 56.5, respectively). No signals of recent population bottlenecks were detected in the brown trout of Lake (sic)ingvallavatn. Discussion: This is the first time that the genetic structure and diversity of brown trout in the watershed of River Olfusa have been assessed. Our results point towards the presence of a metapopulation in the watershed of Lake THORNingvallavatn, which has