We determined quantitative and qualitative alterations in lipids during the occurrence and progression of spinal cord injury (SCI) in rats to identify potential clinical indicators of SCI pathology. Imaging mass spectrometry (IMS) was used to visualize twelve molecular species of phosphatidylcholine (PC) on thin slices of spinal cord with SCI. In addition, twelve species of phospholipids and five species of prostaglandins (PGs) were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) of lipid extracts from control/injured spinal cords. Unique distribution patterns were observed for phospholipids with different fatty acid compositions, and distinct dynamic changes were seen in both their amounts and their distributions in tissue as tissue damage resulting from SCI progressed. In particular, PCs containing docosahexaenoic acid localized to the large nucleus in the anterior horn region at one day post-SCI and rapidly decreased thereafter. In contrast, PCs containing arachidonic acid (AA-PCs) were normally found in the posterior horn region and were intensely and temporarily elevated one week after SCI. Lysophosphatidylcholines (LPCs) also increased at the same SCI stage and in regions with elevated AA-PCs, indicating the release of AA and the production of PGs. Moreover, LC-ESI-MS/MS analysis of lipid extracts from the spinal cord tissue at the impact site demonstrated a peak in PGE2 that reflected the elevation/reduction pattern of AA-PCs and LPC. Although further investigation is required, we suggest that invasive immune cells that penetrated from the impaired blood-brain barrier at 1-2 weeks post-SCI may have produced LPCs, released AA from AA-PCs, and produced PGs in SCI tissue at sites enriched in AA-PCs/LPC.