A fundamental question in ecology is whether microorganisms follow the same patterns as multicellular organisms when it comes to population structure and levels of genetic diversity. Enormous population sizes, predominately asexual reproduction, and presumably high dispersal due to small body size could have profound implications on their genetic diversity and population structure. Here, we have analyzed the population genetic structure in a lake-dwelling microbial eukaryote (dinoflagellate) and tested the hypothesis that there is population genetic differentiation among nearby lake subpopulations. This dinoflagellate occurs in the marine-derived saline lakes of the Vestfold Hills, Antarctica, which are ice-covered most of the year. Clonal strains were isolated from four different lakes, and were genotyped using AFLP (Amplified Fragment Length Polymorphism). Our results show high genetic differentiation among lake populations despite their close geographical proximity (< 9 km). Moreover, genotype diversity was high within populations. Gene flow in this system is clearly limited, either due to physical or biological barriers. Our results discard the null hypothesis that there is free gene flow among protist lake populations. Instead, limnetic protist populations may differentiate genetically, and lakes act as ecological islands even on the microbial scale.