Post-traumatic stress disorder (PTSD) is a widespread psychiatric injury that develops serious life-threatening symptoms like substance abuse, severe depression, cognitive impairments and persistent anxiety. However, the mechanisms of post-traumatic stress injury in brain is poorly understood due to the lack of practical methods to reveal biochemical alterations in various brain regions affected by this type of injury. Here, we introduce a novel method that provides quantitative results from Raman maps in paraventricular nucleus of the thalamus (PVT) region. By means of this approach, we have shown a lipidome comparison in PVT regions of control and PTSD rat brains. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was also employed for validation of the Raman results. Lipid alterations can reveal invaluable information regarding the PTSD mechanisms in affected regions of brain. We have showed that the concentration of cholesterol, cholesteryl palmitate, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, ganglioside, glyceryl tripalmitate and sulfatide changes in the PVT region of PTSD compared to control rats. Higher concentration of cholesterol suggests the higher level of corticosterone in brain.1 Moreover, concentration changes of phospholipids and sphingolipids suggest the alteration of phospholipase A2 (PLA2) which is associated with inflammatory processes in the brain. Our results have broadened the understanding of biomolecular mechanisms for PTSD in PVT region of brain. This is the first report regarding the application of Raman spectroscopy for PTSD studies. This method has a wide spectrum of applications and can be applied to various other brain related disorders or other regions of brain.