Seaweeds are macrophytic algae that have been gaining interest as alternative healthy foods, renewable drug sources, and climate change mitigation agents. In terms of their nutritional value, seaweeds are renowned for their high content of biologically active polyunsaturated fatty acids. However, little is known about the regiochemistry-the geometry and position of carbon-carbon double bonds-of free and conjugated fatty acids in seaweeds. In the present work, a detailed characterization of the seaweed lipidome was achieved based on untargeted HRMS-based analysis and lipid derivatization with a photochemical aza-Paternò-Büchi reaction. A triple-data processing strategy was carried out to achieve high structural detail on the seaweed lipidome, i.e., (i) a first data processing workflow with all samples for aligning peak and statistical analysis that led to the definition of lipid sum compositions (e.g., phosphatidylglycerol (PG) 34:1), (ii) a second data processing workflow in which the samples of each seaweed were processed separately to annotate molecular lipids with known fatty acyl isomerism (e.g., PG 16:0_18:1), and (iii) the annotation of lipid regioisomers following MS/MS annotation of the lipid derivatives obtained following the aza-Paternò-Büchi reaction (e.g., PG 16:0_18:1 ω-9). Once the platform was set up, the lipid extracts from 8 seaweed species from different seaweed families were characterized, describing over 900 different lipid species, and information on the regiochemistry of carbon-carbon double bonds uncovered unknown peculiarities of seaweeds belonging to different families. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of seaweeds.