In a response to gravitropic stress, G-layers (gelatinous layers) were deposited in xylem cell walls of tilted flax plants. G-layers were produced in both tension wood (upper side) as expected but were also observed in opposite wood (lower side). Raman spectral profiles were acquired for xylem G-layers from the tension and opposite side as well as from the G-layer of bast fibers grown under non-tilted conditions. Statistical analysis by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) clearly distinguished bast fiber G-layers from xylem G-layers. Discriminating bands were observed for cellulose (380–1150–1376 cm–1), hemicelluloses (517–1094–1126–1452 cm–1) and aromatics (1270–1599–1658 cm–1). PCA did not allow separation of G-layers from tension/opposite-wood sides. In contrast, the two types of xylem G-layers could be incompletely discriminated through PLS-DA. Overall, the results suggested that while the architecture (polymer spatial distribution) of bast fibers G-layers and xylem G-layers are similar, they should be considered as belonging to a different cell wall layer category based upon ontogenetical and chemical composition parameters.