Seed　germination　plays　a　pivotal　role　in　plant　growth　and　undergoes　many　intricate　biochemical　changes　including　lipid　metabolism.　Nevertheless,　little　is　known　about　lipid　changes　and　distributions　in　different　structures　of　soybean　seeds　during　germination.　Here,　we　applied　mass　spectrometry　imaging　(MSI)　in　conjunction　with　MS-based　lipidomics　to　examine　the　lipid　alterations　in　the　embryo　and　cotyledon　of　soybean　seeds　during　germination.　To　expand　the　coverage　of　lipid　detection　in　soybean　seeds,　we　used　the　novel　techniques　of　matrix-assisted　laser　desorption/ionization　(MALDI)　and　MALDI　coupled　with　laser-postionization　(MALDI-2).　The　results　revealed　that　compared　to　MALDI,　MALDI-2　enhanced　the　detected　numbers　and　intensities　of　lipid　species　in　various　lipid　classes,　except　for　a　few　classes　(e.g.,　sphingomyelin　and　phosphatidylcholine).　Lipidomic　data　showed　that　compared　to　the　embryo,　the　cotyledon　demonstrated　slower　but　similar　lipid　changes　during　germination.　These　changes　included　the　reduced　levels　of　glycerolipids,　phospholipids,　and　sterols,　as　well　as　the　increased　levels　of　lysophospholipids.　Data　from　MALDI&MALDI-2　MSI　supported　and　complemented　these　lipidomic　findings.　Our　work　highlights　the　significance　of　integrating　lipid　profiles　and　distributions　to　enhance　our　understanding　of　the　metabolic　pathways　involved　in　seed　germination.　©　2025