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The diversity of O-polysaccharides (O-antigens) among 28 Aeromonas salmonicida strains isolated from ill fish has been determined by using high-resolution magic angle spinning (HR MAS) NMR spectroscopy. The new O-polysaccharide has been identified in two isolates. This new structure was investigated by 1H and 13C NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The following structure of the linear hexasaccharide repeating unit of A. salmonicida O-antigen has been established: →3)-α-L-Rhap-(1→3)-α-D-ManpNAc-(1→2)-β-D-Glcp-(1→3)-α-L-Rhap2OAc4OAc-(1→3)-β-D-ManpNAc-(1→3)-α-D-Glcp-(1→. This new A. salmonicida O-polysaccharide was detected among two isolates collected from trout and turbot fish in 2010 and 2011, respectively. Further investigations should be conducted to evaluate the distribution of this new O-polysaccharide among a larger collection of isolates, depending on their geographic origin, the species of fish, and the health status of the fish.