Background: Lipid metabolic alterations are recognised as potential oncogenic triggers that promote malignant transformation. Here we performed spatially-resolved profiling of lipid signatures in colorectal cancer (CRC) tissue and matched healthy mucosa using desorption electrospray ionisation imaging mass spectrometry (DESI-MSI). The objectives of this study were to comprehensively define the CRC ‘lipidome’ and to assess lipid signatures in discrete histological regions-of-interest, specifically morphologically bland peri-tumoural epithelium (PT-e) and tumour stroma (T-s). Methods: Fresh frozen tissue sections from 42 patients with confirmed CRC were subjected to negative-ion mode DESI-MSI analysis. Mass spectra in the 200-1000 m/zrange were collated from CRC epithelium (CRC-e), PT-e, T-s and healthy tumour-remote epithelium (TR-e). Spectral signatures were subjected to multivariate analysis using a recursive maximum margin criterion (RMMC) algorithm operating in MATLAB. Results: Increased levels of long/very-long chain fatty acids (LCFA/VLCFA) were seen in CRC-e compared with TR-e(AUC = 0.99). Correspondingly, increased expression of lipogenic and elongase enzymes was found on IHC. Transmission electron microscopy was performed to evaluate peroxisomal distribution and morphology in CRC-e, as these organelles metabolise LCFA/VLCFA through β-oxidation, to negligibly low levels, in healthy cells. No discernible difference in peroxisomal distribution, abundance or structure was found between CRC-e and TR-e. PT-e demonstrated a lipid expression pattern almost identical to that of CRC-e, and markedly different from TR-e (AUC = 0.89). Conclusions: A shift towards increased LCFA/VLCFA production may be an important metabolic trait in CRC facilitated through upregulation of de novo lipogenesis and fatty acid elongation and concurrent impairment of peroxisomal β-oxidation. This phenotype was also observed in morphologically bland PT-e, suggesting that enhanced de novo LCFA/VLCFA biosynthesis and impaired peroxisomal function may represent important steps in peri-tumoural field cancerisation.