Phytoplankton community structure was analysed in the Subantarctic sector of the eastern South Pacific, a high-nutrient, low-chlorophyll (HNLC) region, during a period of deep vertical mixing (austral winter Aug–Oct, 2005) when Antarctic Intermediate Water (AAIW) gets formed, and during a period of water-column stratification characterised by shallow mixed layers (austral summer Feb–Mar, 2006). We examined both the entire phytoplankton assemblage through the use of diagnostic pigments and also the picophytoplankton fraction (<3 μm) through the use of class-specific molecular probes and flow cytometry. Both the pigment and molecular data reveal a dominance of prymnesiophytes during both the winter-mixed and summer-stratified periods. Chrysophytes and their diagnostic pigments were also present throughout most of the study region. Flow cytometric analysis revealed that the contribution of photosynthetic picoeukaryotes (PPEs) to total picophytoplankton (PPEs+picocyanobacteria) abundances increases systematically with increasing latitude and decreasing temperature, highlighting the importance of PPEs in the Southern Ocean.