Naturally-occurring iron nanoparticles constitute a quantitatively-important and biogeochemically-active component of the broader Earth ecosystem. Yet detailed insights into their chemical speciation is sparse compared to the body of work conducted on engineered Fe nanoparticles. The present contribution briefly reviews the analytical approaches that can be used to characterize natural Fe nanoparticles, before detailing a dedicated synchrotron-based X-ray spectro-microscopic investigation into the speciation of suspended Fe nanoparticles collected from fluvial, marine, and lacustrine surface waters. Ferrous, ferric and magnetite classes of Fe nanoparticles (10–100 nm) were identified, and all three classes exhibited a high degree of heterogeneity in the local bonding environment around the Fe center. The heterogeneity is attributed to the possible presence of nanoparticle aggregates, and to the low degrees of crystallinity and ubiquitous presence of impurities (Al and organic moieties) in natural samples. This heterogeneity further precludes a spectroscopic distinction between the Fe nanoparticles and the larger sized Fe-rich particles that were evaluated. The presented results provide an important baseline for natural nanoparticle speciation in pristine aquatic systems, highlight the degree of inter-particle variability, which should be parameterized in future accurate biogeochemical models, and may inform predictions of the fate of released engineered Fe nanoparticles as they evolve and transform in natural systems.