Background: What matter (WM) is particularly sensitive to ischemia and WM changes are observed following onset of ischemic stroke as well as during expansion of the stroke lesion. To better correlate neurobehavioural and functional assessments in our models we have developed imaging methods to aid in the differentiation and quantification of WM injury. Methods: We employ 3 mouse models of stroke: photothrombotic, temporary middle cerebral artery occlusion, and intracerebral hemorrhage. Naïve controls and surgical shams (for each model) are also characterized. We use Fourier transform infrared (FTIR) imaging and synchrotron-based X-ray fluorescence microscopy (XFM) to visualize metabolites and elemental markers, respectively. These post-mortem imaging techniques are combined with conventional histology to confirm neuroanatomic features and cell types. Results: The metabolic profile of WM in naïve, sham, and stroke models has been characterized in C57BL/6 mice. The metabolic markers we identify are highly specific and enable the automated differentiation of WM from other tissues. Our methods have been re-tooled to identify degeneration and injury of WM regions. Conclusions: The combination of FTIR imaging and XFM afford the means to readily differentiate WM changes following stroke onset. Significant dysregulation can be observed before the core or penumbra of the stroke lesion reaches WM-containing regions.