We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed paraffin-embedded (FFPE) human tissue samples. Using high-resolution Matrix-Assisted Laser Desorption/Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry Imaging (MALDI-FT-ICR MSI), we conducted a proof of principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients, and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.