Technical Abstract: Antigenic variation is one mechanism pathogens use to avoid immune-mediated competition between closely related strains. Here, we show that two Bordetella bronchiseptica strains, RB50 and 1289, express two antigenically distinct O-antigen serotypes (O1 and O2 respectively). When 18 additional B. bronchiseptica strains were serotyped, all were found to express either the O1 or O2 serotype. Comparative genomic hybridization (CGH) and PCR screening showed that the expression of either the O1 or O2 serotype correlated with the strain containing either the classical or alternative O-antigen loci respectively. Multi-locus sequence typing (MLST) analysis of 49 B. bronchiseptica strains was used to build a phylogenetic tree, which revealed that the two O-antigen loci did not associate with a particular lineage. This is the first evidence of homologous recombination of an antigenic locus between Bordetella strains. From experiments using mice vaccinated with purified LPS from strain RB50 (O1), 1289 (O2) or RB50'wbm (O-antigen deficient), our data indicate that the protective component of LPS is the O-antigen and that there is a lack of cross protection between LPS vaccines containing different O-antigens. The lack of cross-reaction between O-antigen serotypes also appears to aid in the evasion of antibody-mediated clearance between strains. Our data are consistent with the idea that the O-antigen serotypes of B. bronchiseptica are encoded by separate O-antigen loci that recombine and aid in the evasion of cross-immunity between strains.