Abstract: Sequence analysis and phylogeny of VirD4 protein of Gram-negative bacteria were compared to the classification of the family and knowledge of the evolutionary relationships of its members. Sequences of the gene encoding VirD4 of isolated from Pseudomonas syringae pv. Syringae were used to sequence searches of members of VirD4 protein and putativeVirD4 protein using Basic Local Alignment Search (BLAST). Sequence analysis revealed high amino acid identity among VirD4 proteins. The central region of VirD4 proteins is generally well conserved in comparison to the terminal regions. The phylogenetic analysis was performed among VirD4 proteins and putativeVirD4 protein in 41 species bacteria. In the phylogenetic tree sequences compared were distributed in several well-defined subfamilies. Each subfamily display relationships that are congruent with other subfamilies.