The heartwoods of many wood species have natural resistance to wood decay due to the accumulation of antifungal heartwood extractives. The natural durability of heartwoods has been extensively investigated, yet very little information is available on the initiation of heartwood decay. This experiment examined the onset of Rhodonia placenta brown rot decay in Scots pine heartwood in order to identify the key changes leading to heartwood decay. An imaging approach based on Raman imaging and multivariate image analysis revealed that the degradation of heartwood began in the innermost cell wall layers and then spread into the remaining cell walls and the middle lamella. Pinosylvins were extensively degraded in the cell walls, middle lamella and extractive deposits, while unidentified material most likely consisting of hemicelluloses and/or lipophilic extractives was removed from the inner cell wall layers. Changes similar to inner cell wall degradation were seen in the remaining cell walls in more advanced decay. The results indicate that the key change in incipient heartwood decay is the degradation of antifungal heartwood extractives. The inner cell wall degradation seen in this experiment may serve a nutritive purpose or facilitate the penetration of degradative agents into the cell walls and middle lamella.