Knowledge about the protist diversity of the Pacific sector of the Southern Ocean is scarce. We tested the hypothesis that distinct protist community assemblages characterize large-scale water masses. Therefore, we determined the composition and biogeography of late summer protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea. We used state of the art molecular approaches, such as automated ribosomal intergenic spacer analysis and 454-pyrosequencing, combined with high-performance liquid chromatography pigment analysis to study the protist assemblage. We found distinct biogeographic patterns defined by the environmental conditions in the particular region. Different water masses harbored different microbial communities. In contrast to the Arctic Ocean, picoeukaryotes had minor importance throughout the investigated transect and showed very low contribution south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles were dominating the sequence assemblage in the Subantarctic Zone, whereas the relative abundance of diatoms increased southwards, in the Polar Frontal Zone and Antarctic Zone. South of the Polar Front, most sequences belonged to haptophytes. This study delivers a comprehensive and taxon detailed overview of the protist composition in the investigated area during the austral summer 2010.