MALDI imaging mass spectrometry (MALDI-IMS) has been used successfully in mapping different lipids in tissue sections, yet existing protocols fail to detect the diverse species of mitochondria-unique cardiolipins (CLs) in the brain which are essential for cellular and mitochondrial physiology. We have developed methods enabling the imaging of individual CLs in brain tissue. This was achieved by eliminating ion suppressive effects by (i) cross-linking carboxyl/amino containing molecules on tissue with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and (ii) removing highly abundant phosphatidylcholine head groups via phospholipase C treatment. These treatments allowed the detection of CL species at 100 μm resolution and did not affect the amount or molecular species distribution of brain tissue CLs. When combined with augmented matrix application, these modifications allowed the visualization and mapping of multiple CL species in various regions of the brain including the thalamus, hippocampus, and cortex. Areas such as the dentate and stratum radiatum exhibited higher CL signals than other areas within the hippocampal formation. The habenular nuclear (Hb)/dorsal third ventricle (D3 V) and lateral ventricle (LV) areas were identified as CL "hot spots". Our method also allowed structural MS/MS fragmentation and mapping of CLs with identified fatty acid residues and demonstrated a nonrandom distribution of individual oxidizable (polyunsaturated fatty acid containing) and nonoxidizable (nonpolyunsaturated containing) CLs in different anatomical areas of the brain. To our knowledge, this method is the first label-free approach for molecular mapping of diversified CLs in brain tissue.