Phytoplankton species with a cosmopolitan distribution are traditionally expected to show little genetic differentiation given the lack of geographical barriers in the ocean. To gain an understanding of the genetic variability within southern hemisphere populations of the coccolithophore Emiliania huxleyi, we used eight microsatellite markers to conduct a population genetic analysis on 273 clonal cultures collected from 11 sites in five ocean current systems south of Australia (Leeuwin, Zeehan, East Australian Currents) and in the Southern Ocean, including within the Antarctic Polar Front. Two of the five currently recognised morphotypes were represented, E. huxleyi var. huxleyi and E. huxleyi var. aurorae. Clonality was absent within sampled populations, suggesting the importance of sexual reproduction in the life cycle of this coccolithophore. Significant genetic differentiation (pairwise population FST range = 0.01–0.09) was apparent among E. huxleyi var. huxleyi populations, much higher than for other cosmopolitan plankton species; thus, gene flow between populations must be low. There was evidence of marked differentiation between Southern Ocean populations of E. huxleyi var. aurorae and E. huxleyi var. huxleyi (pairwise FST range = 0.12–0.16), suggesting the existence of a reproductive, environmental and/or biogeographical barrier between these two varieties.