The cotton plant is an essential crop cultivated globally for its fiber and seeds. In this study, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to study the spatial distribution patterns of lipids in cottonseeds. 448 lipid ions were identified by LC-MS/MS, and 24 of which were precisely visualized by using MALDI-MSI. The lipids, including phosphatidylcholines (PC), phosphatidylethanolamines (PE) and triacylglycerols (TG) showed heterogeneous distribution patterns within the cotyledonary and radicle tissues. Additionally, the roles these lipids played in the metabolic pathways were analyzed, and relationship of the spatial distribution of LPC (lysophosphatidylcholine) and corresponding PC was studied. The unique distribution patterns of these lipid metabolites revealed by MSI can provide new insights into areas relating to the spatial compartmentation of lipid metabolism in plants. We believe that the results of MSI, if combined with transcriptomics and proteomics, may offer significant help in genetic engineering work.