Environmental filtering studies have revealed immense oceanic microbial diversity, yet the Southeast Pacific remains comparatively undersampled. We characterize the molecular diversity of phytoplankton across two biogeographic domains with contrasting oceanography—fjords and channels (41–53° S) versus the open Pacific (36–42° S)—where the frequency and intensity of harmful algal blooms (HABs) have increased. Using SSU rRNA metabarcoding, we retrieved community composition and biogeographic patterns for micro-phytoplankton. Diversity signals indicated broadly overlapping communities between domains with subtle shifts along hydrographic and nutrient gradients rather than sharp breaks. Phylogenetic resolution within bloom-forming genera recovered well-supported clades, and multiple ASVs matched historically relevant HAB taxa, including representatives of the Alexandrium complex, Dinophysis, Pseudo-nitzschia, and Karenia. Together, these results suggest that regional environmental filtering acts modestly at the community level while preserving clear signals of taxa of management concern. By providing a regionally resolved, DNA-based baseline for southern Chile’s fjords and adjacent open coast, this study helps fill the molecular diversity gap for the Southeast Pacific and supports improved HAB surveillance and ecosystem forecasting in a climate-sensitive seascape.