Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of 7 Vibrio plasmid replicons. Comparative sequences analysis of the 7 Vibrio plasmid replicons obtained in this study together with 8 published Vibrionaceae plasmid sequences revealed replication participating elements involved in the ColE1-mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation and, as a result the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids has been demonstrated by direct sequencing analysis and southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNAI species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing basis for rapid characterization and classification of Vibrionaceae plasmids.