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Queuosine (Q) is a structurally complex, non‐canonical RNA nucleoside. It is present in many eukaryotic and bacterial species, where it is part of the anticodon loop of certain tRNAs. In higher vertebrates, including humans, two further modified queuosine-derivatives exist ‐ galactosyl‐ (galQ) and mannosyl-queuosine (manQ). The function of these low abundant hypermodified RNA nucleosides remains unknown. While the structure of galQ was elucidated and confirmed by total synthesis, the reported structure of manQ still awaits confirmation. By combining total synthesis and LC-MS-co-injection experiments, together with a metabolic feeding study of labelled hexoses, we show here that the natural compound manQ isolated from mouse liver deviates from the literature-reported structure. Our data show that manQ features an α‐allyl connectivity of its sugar moiety. The yet unidentified glycosylases that attach galactose and mannose to the Q‐base therefore have a maximally different constitutional connectivity preference. Knowing the correct structure of manQ will now pave the way towards further elucidation of its biological function., Mannosyl-queuosine (manQ) is a non-canonical RNA nucleoside present in the anticodon loop of certain tRNAs. Here, the authors use a combination of total synthesis and mass spectrometry to contradict the literature-reported structure and show that manQ features an alpha-allyl connectivity of its mannose moiety.