Introduction: Cerebral ischemia results in a lesion comprised of dying cells surrounded by a band of tissue containing cells at risk of dying, called the penumbra. Understanding the status of energy metabolism in the stroke is necessary to get a clearer picture of the formation of the lesion, and may provide insights into potential therapeutic targets. The detection and distribution of energy metabolism biomarkers, particularly in ischemic brain tissue, presents significant challenges for conventional laboratory techniques due to the labile nature of these markers and the lack of unique chemical or spectroscopic handles. Fourier Transform infrared (FTIR) spectroscopic imaging can be used to map the distribution of different biochemical parameters, such as lipids, proteins, aggregated proteins, glycogen, lactate, pyruvate, ATP/ADP, NADH and glutamate. Methods: We employed the photothrombotic stroke model in adult mice to produce a permanent focal stroke lesion. Brain tissue was collected at multiple time points (1h to 4 weeks post-stroke). FTIR imaging was employed to map the distribution of unique biochemical fingerprints for over a dozen key metabolic markers. Immunohistochemistry was also performed on adjacent tissues to correlate biomarkers revealed by FTIR with the distribution of astrocytes, myelin and macrophages. Results: During the first week poststroke, the lesion shows decreased lipid esters, protein, ATP/ADP, NADH content while showing an increase in glutamate, aggregated protein, lactate and pyruvate. Glycogen can be seen to accumulate in the penumbra starting at 1 day post-stroke until 1 week post-stroke, which correlates with the appearance of astrocytes around the border of the stroke lesion. From 1 week to 4 weeks post-stroke, we observe an increase in lipid esters in the lesion, as well as a decrease in glycogen, lactate and pyruvate, while an increase in ATP/ADP and NADH can be observed. Conclusion: We are showing for the first time novel images of multiple energy metabolism biomarkers across a range of post-stroke time points in a mammalian brain. This type of imaging will be important in studying post-stroke interventions, particularly drugs that are known to alter energy metabolism.