In the present study, antigenic cross-reactivity of OMPs was investigated in several species of bacterial pathogens. Heterogeneous mouse or fish antisera were used to ascertain OMPs with cross-reactivity and cluster analysis was performed to analyze the distribution of cross-antigenic OMPs in diverse bacterial strains. We interestingly found that eleven and seven bands could be reacted with four kinds of heterogeneous mouse and fish antisera, respectively, and the phenograms constructed could provide ideal targeted bacteria for candidate genes of polyvalent vaccines. Importantly, there were significant differences in reaction with bacteria between mouse and fish antisera, but commonly antigenic bands still existed between them. Our results suggest that the cross-reactivity of OMPs exists commonly in Gram-negative bacteria, which may be a promising choice for the development of polyvalent OMP vaccines. Meanwhile, cluster analysis will help to understand the relation of cross-antigenic OMPs among Gram-negative bacteria.