Water is a key element for wood performance, as water molecules interact with the wood structure and affect important material characteristics such as mechanical properties and durability. Understanding wood-water interactions is consequently essential for all applications of wood, including the design of wood materials with improved durability by chemical modification. In this work, we used Raman micro-spectroscopy in combination with a specially designed moisture chamber to map molecular groups in wood cell walls under controlled moisture conditions in the hygroscopic range. We analyzed both untreated and chemically modified (acetylated to achieve two different spatial distributions of acetyl groups within the cell wall) Norway spruce wood. By moisture conditioning the specimens successively to 5, 50, and 95% relative humidity using deuterium oxide (D2O), we localized the moisture in the cell walls as well as distinguished between hydroxyl groups accessible and inaccessible to water. The combination of Raman micro-spectroscopy with a moisturizing system with deuterium oxide allowed unprecedented mapping of wood-water interactions. The results confirm lower moisture uptake in acetylated samples, and furthermore showed that the location of moisture within the cell wall of acetylated wood is linked to the regions where acetylation is less pronounced. The study demonstrates the local effect that targeted acetylation has on moisture uptake in wood cell walls, and introduces a novel experimental set-up for simultaneously exploring sub-micron level wood chemistry and moisture in wood under hygroscopic conditions.