Pantoea agglomerans and other Pantoea species cause infections in humans and are also pathogenic to plants, but the diversity of Pantoea strains and their possible association with hosts and disease remain poorly known, and identification of Pantoea species is difficult. We characterized 36 Pantoea strains, including 28 strains of diverse origins initially identified as P. agglomerans, by multilocus gene sequencing based on six protein-coding genes, by biochemical tests, and by antimicrobial susceptibility testing. Phylogenetic analysis and comparison with other species of Enterobacteriaceae revealed that the genus Pantoea is highly diverse. Most strains initially identified as P. agglomerans by use of API 20E strips belonged to a compact sequence cluster together with the type strain, but other strains belonged to diverse phylogenetic branches corresponding to other species of Pantoea or Enterobacteriaceae and to probable novel species. Biochemical characteristics such as fosfomycin resistance and utilization of D-tartrate could differentiate P. agglomerans from other Pantoea species. All 20 strains of P. agglomerans could be distinguished by multilocus sequence typing, revealing the very high discrimination power of this method for strain typing and population structure in this species, which is subdivided into two phylogenetic groups. PCR detection of the repA gene, associated with pathogenicity in plants, was positive in all clinical strains of P. agglomerans, suggesting that clinical and plant-associated strains do not form distinct populations. We provide a multilocus gene sequencing method that is a powerful tool for Pantoea species delineation and identification and for strain tracking.The genus Pantoea includes several species that are generally associated with plants, either as epiphytes or as pathogens, and some species can cause disease in humans. Pantoea agglomerans, the Pantoea species most commonly isolated from humans, is widely distributed in nature and has been isolated from numerous ecological niches, including plants, water, soil, humans, and animals. It is frequently associated with plants as an epiphyte or an endophyte, and some isolates have been reported to be tumorogenic pathogens (20, 51). As an opportunistic human pathogen, P. agglomerans can occur sporadically or in outbreaks. At the beginning of the 1970s, P. agglomerans (then called Enterobacter agglomerans) was implicated in a large U.S. and Canadian outbreak of septicemia caused by contaminated closures on bottles of infusion fluids; 25 hospitals were involved, with 378 cases (34). Since then, P. agglomerans bacteremia has also been described in association with the contamination of intravenous fluid, parenteral nutrition, the anesthetic agent propofol, blood products, and transference tubes used for intravenous hydration (2-4, 22, 36). P. agglomerans has been recovered from joint fluids of patients with arthritis, synovitis, or osteomyelitis (7). Infection often occurs after injuries with plant thorns, wood slivers, or wooden spl...