One of the most striking ecological divides on Earth is between marine and nearby freshwater environments, as relatively few taxa can move between the two. Microbial eukaryotes contribute to biogeochemical and energy cycling in both fresh and marine waters, with little species overlap between the two ecosystems. Arctic and sub-Arctic marine systems are relatively fresh compared to tropical and temperate systems, but details of microbial eukaryote communities along river-to-sea transitions are poorly known. To bridge this knowledge gap, we investigated three river-to-sea transitions (Nelson, Churchill, and Great Whale Rivers) in sub-Arctic Hudson Bay through 18S rRNA amplicon sequencing to identify microbial eukaryotes along the salinity and biogeochemical gradients. Salinity acted as the principal dispersal barrier preventing freshwater microorganisms from colonizing marine coastal waters, with microbial eukaryote communities of the three rivers clustering together. Just offshore, communities clustered by coastal regions associated with nutrient concentrations. Analysis of indicator species revealed that communities in the nitrate-depleted coastal water off the Churchill and Great Whale Rivers were dominated by heterotrophic taxa and small photosynthetic protists. In contrast, the Nelson offshore community was characterized by a high proportion of the diatom Rhizosolenia. A distinct community of heterotrophic protists was identified in the three estuarine transition zones, suggesting specialized estuarine communities. Such specialization was most marked in the Nelson River system that was sampled more intensely and showed estuarine circulation.The autochthonous community was composed of the bacterial grazers Katablepharis, Mataza, and Cryothecomonas, as well as brackish species of the diatoms Skeletonema and Thalassiosira. These findings suggest that flow regulation on the Nelson River that modifies estuarine circulation would affect estuarine community composition and distribution in the transition zone.