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The Western Tropical North Atlantic is a highly dynamic marine system where the Amazon River Plume (ARP) generates a patchwork of environmental conditions that favor different phytoplankton groups. To study phytoplanktonic community structure in such heterogeneous conditions, we used a set of five standard ship-based measurements taken from oceanographic surveys between 2010 and 2021 to characterize different habitat types. We then utilized a variety of multiparametric approaches to examine phytoplankton biodiversity in the different habitats to assess the biological relevance of our delineated habitats. Our approach generated a consistent set of habitat types across cruises carried out in multiple different years and the Amazon’s two predominant (wet and dry) seasons. Our phytoplankton community analyses revealed strong distinctions among all habitats along the plume gradient usingin-vivofluorescence and diagnostic pigments, and clear contrasts of diazotroph community along the mesohaline waters using direct cell-count, a pattern consistent with niche partitioning among similar species. The few apparent mismatches we found between phytoplankton community composition and habitat may reflect recent hydrographic changes driven by mixing and/or upwelling and thus may be a useful index to biologically-relevant temporal variation. Our habitat classification approach is straightforward and broadly applicable in identifying biologically distinct areas within heterogeneous and dynamic regions of the ocean.