Silica polycondensation occurring in diatom organelles called silica deposition vesicles (SDVs) leads to valve and girdle band formation to complete the biosilica cell wall after cell division. As SDVs have as yet not successfully been isolated, the role of SDV membranes in silica biogenesis is still largely unexplored. Here we used the possibility of synchronizing the diatom Thalassiosira pseudonana to produce two synchronized cultures in different cell cycle stages that differ in their valve SDV production. Lipid subclass fold changes between valve SDV-enriched samples and cells in the G1 state are interpreted as indications for SDV-relevant membrane lipids. The lipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and lysophosphatidylcholine (LPC) were found to be elevated in valve SDV-enriched cells, also showing accumulation of the very long-chain polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). It is conceivable that PA and LPC are preferentially found in SDV regions with high membrane curvature like the rims of the pancake-shaped valve SDV, while PC and PE molecules are presumably found in the planar regions of the SDVs. In addition, DHA-enriched phospholipids are known to form highly disordered membrane domains, which might be involved in membrane protein localization, possibly used for cytoskeleton-mediated positioning of the SDV.