The chemical heterogeneity (charge distribution) of polyamide reverse osmosis (RO) membrane surfaces and its influence on membrane fouling were investigated using atomic force microscopy (AFM). Negatively charged carboxyl modified latex (CML) and positively charged aliphatic amine latex (AAL) particles were employed to make AFM colloidal probes. The adhesive forces were significantly higher with the AAL probe due to negatively charged membrane surfaces. The chemical heterogeneity measured by AFM colloidal probes indicated that RO membranes could have chemically heterogeneous surfaces even if they exhibited similar average surface properties including zeta potential, contact angle and surface roughness. By performing fouling experiments with two commercial RO membranes of different chemical heterogeneities, it was demonstrated that RO membrane with high chemical heterogeneity was more prone to fouling, suggesting that surface charge distribution is one of the key factors governing RO membrane fouling. Consequently, surface chemical heterogeneity provides a new tool for accessing membrane fouling mechanisms and a novel concept for developing fouling resistant RO membranes. (C) 2015 Elsevier B.V. All rights reserved.