Tension wood is produced on the upper sides of the inclined trunk or branch of hardwood when its orientation is shifted from the vertical. Unlike the opposite wood formed on the lower sides, tension wood fiber is characterized by the presence of a specific layer, called the gelatinous layer which displays various physical and chemical properties. In this study, TEM imaging was used to reveal the variation in the cell wall layering structure between the Populus nigra tension wood and opposite wood. Furthermore, confocal Raman microscopy with 532 nm exciting laser (spatial resolution is about 0.5 μm) was used to visualize the variation in the distribution of fiber wall components and porosity between tension wood and opposite wood, meanwhile the topochemical correlation was innovatively revealed by Raman overlaid image. When integrating over the Raman band at 2 942 cm-1 (Cellulose, hemicelluloses and lignin C—H stretching vibration), the sublayers of tension wood and opposite wood were successfully distinguished. By integrating over the band at 1 094, 1 598 and 904 cm-1 in the normalized average Raman spectra, the distribution of cellulose, lignin and xylan was visualized. Lignin-cellulose and lignin-xylan overlaid Raman images displayed that for the tension wood cellulose mainly existed in the gelatinous layer, and the concentration of cellulose and xylan within the cell wall regions was higher than that of opposite wood. Specifically, the secondary wall of tension wood had an increased lignin concentration. Double-wall line scan showed that the distribution of lignin, cellulose and xylan was highly regional dependence and displayed gradient changes along the adjacent cell wall. Moreover, line scan analysis revealed the variation in D2O concentration along the adjacent fiber secondary wall and confirmed the more abundant porosity distribution of the gelatinous layer compared with secondary wall and middle lamella. The above results are helpful to understand the forming mechanism of unique physical and chemical properties of tension wood and expand the application of micro Raman-spectroscopy technology in the field of plant cell wall pore structure research.