Genome sequencing reveals that three non-flagellar (PAI1, PAI2 and PAI3) and two flagellar (Fla1 and Fla2) type III secretion systems (T3SSs) exist in the genomes of Erwinia amylovora strains Ea273 and CFBP1430. In this study, our goal was to determine the origin as well as potential function of T3SSs in E. amylovora. The phylogenetic tree constructed using conserved HrcV or InvA protein indicated that the three non-flagellar T3SSs belong to the Hrp1 and Inv/Spa groups, respectively. The concatenated phylogenetic tree constructed from 14 conserved flagellar proteins suggested that both flagellar systems were originated from the enterobacterial branch of the gamma-proteobacteria. Interestingly, among these phylogenetic trees, one clade each containing non-flagellar (PAI2 and PAI3) and the tightly-clustered flagellar (Fla2) T3SSs, respectively, was both clustered together with those from Sodalis glossinidius, suggesting PAI2, PAI3 and Fla2 may be acquired from a similar unknown source. We have previously demonstrated that the PAI2 and PAI3 are not involved in virulence on host plants. Gene expression analysis revealed that the regulatory genes of the PAI2 and PAI3 were differentially expressed in rich medium at high temperature (°), in contrast to those reported for PAI1, which expresses in minimum medium at low temperature (20°C). In addition, we generated deletion mutants of Fla1 and Fla2, and their effect on flagella biosynthesis and swarming motility were also examined. Our results showed that deletion of the whole Fla2 system did not affect swarming motility and flagellar formation; whereas deletion of a single flhDC1 or fliA1 gene in Fla1 system completely abolished flagellar formation and swarming motility. These results suggest that PAI2, PAI3 and Fla2 might function in other hosts of E. amylovora, other than the plant host.