Three housekeeping genes (dnaK, groEL and rpoB) of strains belonging to the genus Gluconacetobacter (37 strains) or related taxa (38 strains) were sequenced. Reference strains of the 15 species of the genus Gluconacetobacter were included. Phylogenetic trees generated using these gene sequences confirmed the existence of two phylogenetic groups within the genus Gluconacetobacter. These groups clustered separately in trees constructed using concatenated sequences of the three genes, indicating that the genus Gluconacetobacter should not remain a single genus and should be split, as suggested previously. Multilocus sequence analysis (MLSA) of the three housekeeping genes also proved useful for species differentiation in the family Acetobacteraceae. It also suggested that Gluconacetobacter xylinus LMG 18788, better known as the type and only strain of Acetobacter xylinus subsp. sucrofermentans, represents a distinct species in the genus Gluconacetobacter, and is not a true G. xylinus strain. In previous studies, this strain showed less than 70 % DNA relatedness to the type strains of G. xylinus and Gluconacetobacter nataicola, the phylogenetically nearest relatives, and could be distinguished from them phenotypically. Additionally, AFLP and (GTG)(5)-PCR DNA fingerprinting data supported its reclassification within a distinct species. The name Gluconacetobacter sucrofermentans (Toyosaki et al. 1996) sp. nov., comb. nov. is proposed.