Understanding phytoplankton assemblages in reef waters is vital to comprehend their spatial variation and overall productivity. However, global research on these assemblages remains limited, especially using pigment-based high-performance liquid chromatography (HPLC)-CHEMTAX approach. Therefore, in this study, we compare the phytoplankton assemblages in the Southwestern Atlantic, including reef areas and coastal reefless areas, using the HPLC-CHEMTAX method. Water column analysis revealed well‑oxygenated conditions, with euphotic zones extending from 8.6 up to 15.1 m. Similarities in abiotic variables between surface and mid-water samples indicated a thoroughly mixed water column. Phosphate and dissolved inorganic nitrogen (DIN) concentrations remained low, ranging from ≤0.14 to 0.64 and ≤ 0.10 to 1.51 μM, respectively. Dissolved reactive silicon (DSi) concentrations varied from ≤0.14 to 23.12 μM, with high concentrations observed in coastal samples. Overall, phytoplankton biomass was modest, with total chlorophyll a levels ranging from 0.18 to 0.91 mg m−3. Diatoms, prymnesiophytes, and picocyanobacteria accounted for 91.2 % of the total biomass, whereas green flagellates, dinoflagellates, and cryptophytes made minor contributions. Phytoplankton composition differed between reefs and coastal areas, but no significant differences were found in the total biomass. Diatoms were prevalent, especially near the reefs, where a decrease in silica concentrations was observed. In contrast, Synechococcus and prymnesiophytes contributed more nearshore and beyond the reef areas. The distinct phytoplankton composition near reefs, even in oligotrophic waters, underscores the important role of tropical reefs in shaping phytoplankton assemblages via benthic–pelagic coupling. © 2024 Elsevier B.V.