Exploring the molecular aspects of the human brain’s structural arrangement can provide insights into its underlying functional mechanisms. The search for differences in white matter regions often focuses on measuring myelin layer thicknesses or parameters of diffusion tensor imaging. Lipids are vital components of cell membranes and are involved in important physiological functions such as signal transmission, maintaining membrane structure, and supporting the development of neurons. This research involved analyzing the distribution of lipids in different areas of the human brain, specifically in the gray matter of the neocortex and two white matter regions representing the cingulum bundle and corpus callosum axonal tracks. Method of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used for discovering lipid composition differences not only between gray and white matter, but also between two axonal tracks. Using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis of these brain regions, we further identified specific lipid classes that distinguished the two white matter regions. Thus, corpus callosum was enriched with MGDG, NAE, Cer and PC-O lipid classes, while cingulum bundle is enriched CL, PG, LPE and PI classes. A variation in fatty acid composition was noted: lipids in the cingulum bundle had shorter fatty acids, while those in the corpus callosum had longer fatty acids. Comparison of lipid profiles of gray and white matter coincided with literature data. To sum up, our findings show that each axonal pathway has a specific molecular organization that matches what we learn from studying functional brain networks using MRI.