Extracellular vesicles (EVs) are highly specialized, nanoscale messengers that deliver biological signals and in doing so mediate intercellular communication. Increasing evidence shows that within populations of EVs, important properties including morphology, membrane composition, and content vary substantially. This heterogeneity arises in response to the nature, state, and environmental conditions of the cell source. However, currently there are no effective approaches, which unequivocally discriminate differences between individual EVs, which critically hampers progress in this emerging scientific area. Measuring EV heterogeneity is paramount to our understanding of how EVs influence the physiological and pathological functions of their target cells. Moreover, understanding EV heterogeneity is essential for their application as diagnostics and therapeutics. We propose an innovative approach using resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR) to identify the nanoscale structural composition of EVs, as demonstrated and validated using EVs derived from two types of placenta stem cells. The particular strength of this approach is that it is a label-free and ultra-high sensitivity technique that has the power to measure individual EV heterogeneity. New insights gained by this method into EV heterogeneity will have a profound impact not only on our basic understanding of EV biology but also on disease diagnostics and the emerging area of EV-therapies.