The objectives of this thesis were the establishment of molecular approaches in the diversity investigation of eukaryotic protists in the Southern Ocean, the comparison of different approaches and the delivery of a comprehensive and taxon detailed overview of protist assemblages in the Pacific sector of the Southern Ocean, especially in the Amundsen Sea. The molecular approaches used to achieve these goals were automated ribosomal intergenic spacer analysis (ARISA), sequencing of 18S rRNA gene clone libraries and 454-pyrosequencing. The comparison of 18S rRNA clone library sequences with the results of 454-pyrosequencing was conducted with four Arctic water samples, focusing on picoplankton (0.4-3 μm), and with one Antarctic water sample, covering the whole size spectrum (>0.2 μm). It turned out that the two methods delivered different results. Both approaches discovered phylotypes that were not found with the other approach. The abundant biosphere, defined by the 454-pyrosequencing approach, was not fully recovered by the clone library approach. The cloning approach was biased against several groups, e.g. haptophytes in the Arctic samples and diatoms in the Antarctic sample. In summary, prior cloning data have to be handled with care, when compared with 454-pyrosequencing data. Additionally, cloning data are only of limited suitability to serve as a backbone for phylogenetic analysis of 454-pyrosequencing data. The results of this comparison led to the decision to use ARISA and 454-pyrosequencing for the further protist diversity investigations. First, the hypothesis that distinct protist community assemblages characterize large-scale water masses was tested. The composition and biogeography of late summer eukaryotic protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea was determined. ARISA and 454-pyrosequencing were used in combination with flow cytometry and pigment measurements via high performance liquid chromatography (HPLC) to study the protist assemblage. Distinct biogeographic patterns defined by the different oceanic regions were revealed. Different water masses harboured different microbial communities, and environmental gradients limited their dispersal. Picoeukaryotes were of minor importance throughout the investigated transect and were nearly absent south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles dominated the Subantarctic Zone, whereas the importance of diatoms increased southwards, in the Polar Frontal Zone, the Antarctic Zone and the Subpolar Region. South of the Polar Front, haptophytes were the dominating group. Second, the investigation focused on the Amundsen Sea to see if the protist community assemblages vary in different areas of a single large-scale water mass. The composition and structure of late summer eukaryotic protist assemblages along a west-east transect in the Amundsen Sea were analysed. ARISA and 454-pyrosequencing were combined with HPLC. Characteristic communities offshore and inshore were revealed, but the differences were weaker, compared to those found along the north-south transect. In general, total chlorophyll a and microeukaryotic contribution were higher in inshore samples. Picoeukaryotes were also of minor importance. Diatoms were the dominating group across the entire area, at which Eucampia sp. and Pseudo-nitzschia sp. were dominating inshore and Chaetoceros sp. was dominating offshore. At the eastern most station, the assemblage was dominated by Phaeocystis sp. Under the ice, ciliates showed their highest and haptophytes their lowest abundance. This thesis sheds light on the use and applicability of several molecular methods for the investigation of protist assemblages in polar waters. It delivers a comprehensive and taxon detailed overview of the eukaryotic protist composition during the austral summer in the Pacific sector of the Southern Ocean, especially in the Amundsen Sea. This thesis constitutes as groundwork for future investigations of protist assemblage changes in this area.