Microalgae are only a few micrometres to a millimetre in size, yet they constitute the base for aquatic food webs and are extremely important drivers in elemental cycling. Long it has been assumed that small organisms (<1 mm) that occur in high abundances, have global dispersal potential facilitated by winds, currents and vectors e.g. birds and insects. A growing body of evidence is however portraying a different story; that several planktonic microalgal species exhibit genetically differentiated populations at regional geographic scales, and that populations can also differ in environmentally important phenotypic traits. In this thesis I present results from field studies and experiments designed to test various physical and biological dispersal barriers to explain why, despite the high dispersal potential, microalgae exhibit reduced gene flow (exchange of genetic material) between populations. A marine species Skeletonema marinoi (Bacillariophyceae) and a freshwater species Gonyostomum semen (Raphidophyceae) were used to test the function of geographic distance and hydrographic connectivity (physical barriers) as well as local adaptation and priority effects (biological barriers) on gene flow between populations. Geographic distance and oceanographic connectivity (currents) have demonstrated suitable predictors for mapping gene flow between populations of various marine microalgae species. Indeed, we also found that over large (>1000 km) regions in the Baltic Sea, population genetic patterns could be correlated with dispersal limitation caused by geographic distance and oceanographic connectivity. Additionally, when genetic data was compared to environmental data, population differentiation was corroborated by differences in salinity and silicate concentrations, indicative of adaptation as a driver of population genetic divergence. Over smaller (<160 km) distances, although reduced gene flow was evident, physical dispersal barriers were poor predictors of local population genetic patterns. Surprisingly, the role of connectivity by currents (marine systems) and rivers (freshwater systems) did not show any signs of facilitating dispersal that resulted in gene flow. In the freshwater study area, we found a weak effect of geographic distance, whereas in the marine study, neither distance nor currents could explain signals of reduced gene flow, suggesting other influences driving population genetic differentiation, e.g. environmental selection against immigrants or persistent historical events reinforced by adaptation (monopolization). Laboratory experiments using strains of S. marinoi confirmed that prior arrival to a vacant resource (founder event) was associated with a competitive advantage compared to later arriving strains (priority effects). In addition, strains were competitively superior in native salinity conditions, compared to non-native strains, supportive of local adaptation. Combined these results provide strong support for monopolization (priority effects reinforced by adaptation) as an important biological barrier to gene flow between nearby populations of planktonic microalgae. In summary, this thesis provides important insight into the physical and biological barriers that act in concert in driving population genetic differentiation and diversification of these ecologically central actors. In addition, from an evolutionary perspective these results highlight that the history and biology of these organisms are equally important features to consider as well as their physical characteristics that may facilitate dispersal.