Hypothesis: Molecular dynamics simulation can be used to differentiate between the adsorption proper ties of rhamnolipid congeners at a vacuum-water interface. Experiments: Adsorption of five congeners with differing alkyl chains (two C10 chains, two C14 chains or mixed C14C10 and C10C14), number of rhamnose rings (monoor di-) and carboxyl group charge (non-ionic or anionic) are simulated at the vacuum-water interface. Findings: All rhamnolipids adsorb in the interfacial region with rhamnose and carboxyl groups closer to the water phase, and alkyl chains closer to the vacuum phase, but with differing adsorbed conformations. Headgroups of uncharged congeners show two preferred conformations, closed and partially open. Dirhamnolipid has a low proportion of closed conformation, due to the steric constraints of the second pyranose ring. Charged congeners show strong preference for closed headgroup conformations. For rhamnolipids with equal alkyl chains lengths (C10C10, C14C14) the distribution of alkyl chain tilt angles is similar for both. Where chain lengths are unequal (C14C10, C10C14) one chain has a greater tendency to tilt towards the water phase (>90 degrees). The order parameter of the alkyl chains shows they are disordered at the interface. Together, these results show congener-dependent adsorbed conformation differences suggesting they will have differing surface-active properties at vacuum-water and oil-water interfaces. (C) 2020 The Author(s). Published by Elsevier Inc.