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The Southwest Atlantic Ocean (SWAO) is a spatially dynamic region with a remarkably high primary productivity. An exhaustive identification of the members of its phytoplankton community is a key step to understand the processes that sustain this ecosystem. Here, we provide a community composition analysis of eukaryotic phytoplankton in four SWAO sectors. We gathered 18S rRNA gene metabarcoding data and complemented it with confocal microscopy images, both from the Tara Oceans expedition in late spring 2010. Our work showed local and regional variation across three different size fractions that reflect the complexity of this region. Diversity decreased along temperature and latitudinal gradients, but also showed intricate patterns of occurrences across samples, suggesting that multiple factors shape the community structure. Samples resembled communities from other temperate regions and showed an increasing influence by cold waters from the Southern Ocean towards higher latitudes. These results complement previous regional studies that used other methods such as microscopy or pigment analysis. Our study contributes to the beginning of genomic-based surveys of plankton communities in the SW Atlantic and calls for further work in the region to enhance ecosystem monitoring and projections in the context of global change. [ABSTRACT FROM AUTHOR]