Acute injury is one of the substantial stage post-traumatic brain injury (TBI) occurring at the moment of impact. Decreased metabolism, unregulated cerebral blood flow and direct tissue damage are triggered by acute injury. Understating the biochemical alterations associated with acute TBI is critical for brain plasticity and recovery. The objective of this study was to investigate the biochemical and molecular changes in hippocampus, corpus callosum and thalamus brain regions post-acute TBI in rats. Fourier Transform Infrared (FTIR) imaging spectroscopy were used to collect chemical images from control and 3 hrs post-TBI (Marmarou model was used for the TBI induction) rat brains and adjacent sections were treated by hematoxylin and eosin (H&E) staining to correlate with the disruption in tissue morphology and injured brain biochemistry. Our results revealed that the total lipid and total protein content decreased significantly in the hippocampus, corpus callosum and thalamus after brain injury. Reduction in lipid acyl chains (–CH2) associated with an increase in methyl (–CH3) and unsaturated lipids olefin = CH concentrations is observed. Furthermore, there is a decrease in the lipid order (disorder), which leads to an increase in acyl chain fluidity in injured rats. The results suggest acute TBI damages brain tissues mechanically rather than chemical alterations. This will help in assessing successful therapeutic strategy in order to mitigate tissue damage in acute TBI period.