Fragmentation of habitats is one of the major threats to biodiversity. Fragmentations will reduce connectivity and consequently affect the genetic population structure particularly in rivers. In the current study the genetic population structure of grayling Thymallus thymallus in a large Norwegian river system with both unobstructed and fragmented sections was investegated. Further, it was assessed whether grayling exhibited homing and thereby were genetically structured within the large unobstructed river section of the river system in question. This was done by using telemetry data of 144 radio tagged grayling, to assign individuals in specific spawning locations. Genotyping data on 12 microsatellite markers was then used to infer, the genetic structure of the individuals from the different spawning locations. To infer the effects of migration barriers on genetic population structure, the population structure in the whole river system was investigated, which contained both a natural and anthropogenic migration barrier. No genetic structuring for grayling in relation to spawning fidelity in the unobstructed river section was found. This may indicate that grayling have no precise homing to particular spawning locations and probably move among different spawning habitats in the river section throughout their lives. Four genetically distinct populations in the whole river system was found, which seemed to be separated by the migration barriers. The migration barriers seemed also to direct the gene flow downstream, which have lead to the highest genetic diversity being found downstream. The anthropogenic migration barrier gave indications of acting as a stronger migration barrier to downstream gene flow compared to the natural migration barrier. Identifying present genetic structure as a consequence of geographic isolation or the use of different spawning habitats is important for understanding and retaining connectivity for fish in rivers.