<p>Human pancreatic islets are highly heterogeneous; thus, understanding their biological organization is crucial for elucidating metabolic function and diabetes pathogenesis. High-resolution mass spectrometry imaging of intact human pancreas is challenging due to the small size and dispersed distribution of individual islets within dense exocrine tissue. Here, we establish an ultra-low-flow-rate DESI mass spectrometry imaging (u-DESI-MSI) platform that enables lipidomic analysis of individual islets in human pancreatic tissue. Optimization of raster step size and scan rate parameters for u-DESI resulted in lipid ion images with enhanced spatial fidelity. The analysis demonstrates a highly reproducible central–peripheral spatial lipid distribution within pancreatic tissue. Diacyl phosphatidylcholines (diacyl-PCs), ether-linked phosphatidylcholines (ether-linked PCs) and sphingomyelins (SMs) are predominantly localized to the central endocrine region and co-register with intact insulin distributions, as further validated by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). In contrast, specific lysophosphatidylcholines (LPCs) delineate the endocrine–exocrine interface. These results indicate the existence of position-dependent lipid organization within human islets. This platform provides a robust spatial-mapping foundation for future studies investigating structural and metabolic alterations in human islets under disease conditions.</p> Graphical abstract <p></p>