Cellulose nanocrystals (CNC) are the focus of significant attention in the broad area of sustainable technologies for possessing many desirable properties such as a large surface area, high strength and stiffness, outstanding colloidal stability, excellent biocompatibility and biodegradability, low weight and abundance in nature. Yet, a fundamental understanding of the micro- and nanoscale electrical charge distribution on nanocellulose still remains elusive. Here we present direct quantification and mapping of surface charges on CNCs at ambient condition using advanced surface probe microscopy techniques such as Kelvin probe force microscopy (KPFM), electrostatic force microscopy (EFM) and force-distance (F-D) curve measurements. We show by EFM measurements that the surface charge in the solid-state (as contrasted with liquid dispersions) present at ambient condition on CNCs provided by Innotech Alberta is intrinsically negative and the charge density is estimated to be 13 μC/cm. These charges also result in CNCs having two times the adhesive force exhibited by SiO substrates in adhesion mapping studies. The origin of negative surface charge is likely due to the formation of CNCs through sulfuric acid hydrolysis where sulfate half esters groups remained on the surface (Johnston et al., 2018).