Traumatic brain injury (TBI) is a major cause of death and disability in children and young adults worldwide according to the World Health Organization (WHO). The emergence of mass-spectrometry-based techniques, such as MALDI-MSI, has allowed the monitoring and visualization of changes post injury, providing a global picture of the impact of TBI on different classes of molecules in a single study. In this work, we show the ability to track lipid changes post-TBI by three-dimensional matrix-assisted laser desorption/ionization-mass-spectrometry imaging (MALDI-MSI). Controlled cortical impact (CCI) was induced on adult male rats resulting in direct mechanical injury to the cortical tissue on the right ipsilateral hemisphere of the brain. Images of lipid distribution in coronally sectioned injured brains were acquired using a high-resolution mass spectrometer (MALDI-LTQ-Orbitrap-XL). Results reveal unique lipid signatures for the injured cortical tissue, which further segregate into two subgroups of injury (lesion interior and lesion exterior). Although both subgroups show different profiles from that of the noninjured cortical tissue, the lesion interior is more similar to the ventricular system than the lesion exterior. For example, m/ z 725.56 showed expression in both injured tissue and the ventricular system, whereas m/ z 856.59 (phosphatidylcholine 42:9) is uniquely expressed in injured tissue. On the other hand, m/ z 797.59 (also a phosphatidylcholine) showed unique expression to the ventricular system and not to the injured cortical tissue. Our results can help in further monitoring and identifying lesion-specific lipids in a 3D manner to obtain a better understanding and visualization of molecular and cellular events occurring post-TBI.