Microscopic organisms are widespread and many species have a cosmopolitan distribution. This pattern suggests that microorganisms, in contrast to most larger organisms, have unlimited dispersal. Unlimited dispersal implies high migration rates and gene flow within a species, and consequently no opportunity for populations to differentiate. However, in cosmopolitan microeukaryotes, many studies show that populations are genetically highly differentiated. This implies limited gene flow despite high dispersal, posing a dispersal–differentiation paradox. Within this framework we are investigating population genetic structure and bottlenecks in the freshwater species Chlamydomonas. The aim is to determine if the population consist of multiple subpopulations and if it undergoes population bottlenecks. Here we have sequenced the genomes of approx 90 individual strains from a single lake population across two years. Our aim is to identify SNPs to perform downstream population genetic analysis including estimates of effective population size.