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Abstract A mouse monoclonal antibody (mAb FL100A) previously prepared againstFlavobacterium psychrophilum(Fp) CSF259‐93 has now been examined for binding to lipopolysaccharides (LPS) of this strain andFp950106‐1/1. The corresponding O‐polysaccharides (O‐PS) of these strains are formed by identical trisaccharide repeats composed ofl‐Rhamnose (l‐Rha), 2‐acetamido‐2‐deoxy‐l‐fucose (l‐FucNAc) and 2‐acetamido‐4‐R₁‐2,4‐dideoxy‐d‐quinovose (d‐Qui2NAc4NR₁) where R₁represents a dihydroxyhexanamido moiety. The O‐PS loci of these strains are also identical except for the gene (wzy1orwzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O‐PS repeats are joined throughd‐Qui2NAc4NR₁andl‐Rha bywzy2‐dependent α(1–2) linkages inFpCSF259‐93 versuswzy1‐dependent β(1–3) linkages inFp950106‐1/1. mAb FL100A reacted strongly withFpCSF259‐93 O‐PS and LPS but weakly or not at all withFp950106‐1/1 LPS and O‐PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5‐times stronger to homologousFpCSF259‐93 LPS than to LPS from a strain with a different R‐group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O‐PS. Thus,FpCSF259‐93 O‐PS formed a stable well‐defined compact helix in which the R₁groups were displayed in a regular pattern on the helix exterior while unreactiveFp950106‐1/1 O‐PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2‐linkedF. psychrophilumO‐PS and LPS.