Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.