Species belonging to the genus of Pantoea are commonly isolated from plants, humans and the natural environment. The species of the genus are phenotypically closely related, making rapid identification of Pantoea strains to the species level difficult. Multilocus sequence analysis (MLSA) was evaluated as a means for rapid classification and identification of Pantoea strains. Four housekeeping genes, gyrB, rpoB, atpD and infB, were sequenced for strains assigned to the genus. Included in the study were (1) reference strains from the seven currently recognized species of Pantoea, (2) strains belonging to Brenner DNA groups II, IV and V, previously isolated from clinical samples and difficult to identify because of high phenotypic similarity to P. agglomerans or P. ananatis and (3) isolates from diseased Eucalyptus, maize and onion, assigned to the genus on the basis of phenotypic tests. Phylogenetic trees were constructed from the sequences of the four housekeeping genes. The ''core'' Pantoea species formed a cluster separate from the ''Japanese'' species which formed a tight cluster that included the genus Tatumella when the tree was based on concatenated sequences of the four genes. The MLSA data further suggested the existence of ten potential novel species, phylogenetically related to the currently recognized Pantoea species and the possible inclusion of Pectobacterium cypripedii in the genus Pantoea. When compared with DNA-DNA hybridization data, a good congruence was observed between both methods, with gyrB sequence data being the most consistent. In conclusion, MLSA of partial nucleotide sequences of the genes gyrB, rpoB, atpD and infB can be used for classification, identification and phylogenetic analyses of Pantoea strains. r 2008 Elsevier GmbH. All rights reserved. The GenBank/EMBL accession numbers for the sequences presented in this study are: EF988753-EF988838, EU145260-EU145275, EU344757-EU344760, FJ187830-FJ187834 (gyrB gene); EF988925-EF989010, EU145292-EU145307, EU344765-EU344768, FJ187840-FJ187844 (rpoB gene); EF988667-EF988752, EU145244-EU145259, EU344753-EU344756, FJ187825-FJ187829 (atpD gene); EF988839-EF988924, EU145276-EU145291, EU344761-EU344764, FJ187835-FJ187839 (infB gene) and EF688006-EF688012, EU216734-EU216737, EU344769-EU344770 (16S rRNA).Ã Corresponding author. Tel.: +2712 420 3934; fax: +2712 420 3960.E-mail address: teresa.coutinho@fabi.up.ac.za (T. Coutinho).Please cite this article as: C. Brady, et al., Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis