Little is known about the underlying basis of serotype specificity among strains of , the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the -polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of 950106-1/1, which belongs to a different O-serotype, are identical except for , which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for CSF259-93 versus β1-3 for 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti- antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of CSF259-93 or 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.