This paper presents research on the genetic structure and diversity of populations of a common marine protist and their changes over time. The bloom-forming diatom Skeletonema marinoi was used as a model organism. Strains were revived from anoxic discrete layers of a 210Pb-dated sediment core accumulated over more than 100 y, corresponding to >40,000 diatom mitotic generations. The sediment core was sampled from the highly eutrophic Mariager Fjord in Denmark. The genetic structure of S. marinoi was examined using microsatellite markers, enabling exploration of changes through time and of the effect of environmental fluctuations. The results showed a stable population structure among and within the examined sediment layers, and a similar genetic structure has been maintained over thousands of generations. However, established populations from inside the fjord were highly differentiated from open-sea populations. Despite constant water exchange and influx of potential colonizers into the fjord, the populations do not mix. One fjord population, accumulated in 1980, was significantly differentiated from the other groups of strains isolated from the fjord. This differentiation could have resulted from the status of Mariager Fjord, which was considered hypereutrophic, around 1980. There was no significant genetic difference between pre- and posteutrophication groups of strains. Our data show that dispersal potential and generation time do not have a large impact on the genetic structuring of the populations investigated here. Instead, the environmental conditions, such as the extreme eutrophication of the Mariager Fjord, are deemed more important.