This study investigates the genetic structure of an eukaryotic microorganism, the toxic dinoflagellate Alexandrium ostenfeldii, from the Baltic Sea, a geologically young and ecologically marginal brackish water estuary which is predicted to support evolution of distinct, genetically impoverished lineages of marine macroorganisms. Analyses of the internal transcribed spacer (ITS) sequences and Amplified Fragment Length Polymorphism (AFLP) of 84 A. ostenfeldii isolates from five different Baltic locations and multiple external sites revealed that Baltic A. ostenfeldii is phylogenetically differentiated from other lineages of the species and micro-geographically fragmented within the Baltic Sea. Significant genetic differentiation (FST) between northern and southern locations was correlated to geographical distance. However, instead of discrete genetic units or continuous genetic differentiation, the analysis of population structure suggests a complex and partially hierarchic pattern of genetic differentiation. The observed pattern suggests that initial colonization was followed by local differentiation and varying degrees of dispersal, most likely depending on local habitat conditions and prevailing current systems separating the Baltic Sea populations. Local subpopulations generally exhibited low levels of overall gene diversity. Association analysis suggests predominately asexual reproduction most likely accompanied by frequency shifts of clonal lineages during planktonic growth. Our results indicate that the general pattern of genetic differentiation and reduced genetic diversity of Baltic populations found in large organisms also applies to microscopic eukaryotic organisms. © 2012 Tahvanainen et al.