To better understand the high recalcitrance of bamboo during bioconversion, the fine spatial distribution of polymers in bamboo was studied with Imaging FTIR microscopy under both transmission and ATR modes, combined with PCA data processing. The results demonstrated that lignin, xylan and hydroxycinnamic acid (HCA) were more concentrated in the fibers near the xylem conduit, while cellulose was evenly distributed across the whole fiber sheath. PCA processing produced a clear separation between bamboo fibers and parenchyma cells, indicating that the parenchyma cells contains more pectin and HCA than fibers. It also demonstrated that cellulose, xylan and S-lignin were concentrated most heavily in bamboo fiber secondary cell walls, while G-lignin, pectin and HCA were found more in the compound middle lamella. The revealed information regarding polymer distribution is of great significance for better understanding of the inherent design mechanism of plant cell wall and its efficient utilization.