Pine Pitch Canker (PPC), caused by the fungus Fusarium circinatum, is associated to significant economic and ecological losses worldwide. The effectiveness of PPC monitoring, early detection in nurseries and plantations, and the identification of resistant plant material relies on the development of objective, non-destructive and cost-effective tools. This study analysed the potential of employing Raman Spectroscopy (RS) for the early detection of biochemical changes associated with PPC in Pinus spp. with different susceptibilities to F. circinatum (highly susceptible Pinus radiata vs. relatively-resistant Pinus pinea), while unveiling possible mechanisms of action on these pathosystems. Our results indicate lignin as a key constitutive component of pine resistance against PPC and thus the potential of using this technology for the selection of PPC resistant trees. Moreover, we demonstrate the power of RS-based approaches for the rapid detection of the disease in susceptible species. Early spectral variations were found in P. radiata upon inoculation with F. circinatum from 3 days post-inoculation (dpi) onwards, whereas changes in histological analysis, relative internal stem necrosis measurements, and visual disease symptoms were only visible at 6, 8, and 9-dpi, respectively. These spectral changes have been associated to cell-wall degradation and induction of phenolic compounds synthesis upon infection in P. radiata. Altogether, we believe that RS is an innovative promising tool able to reduce disease detection time in pine species and providing an appealing alternative for the development of new and eco-friendly disease control measures.