Resequencing of the 16S rRNA gene of the type strain of Herbaspirillum putei Ding and Yokota 2004 revealed 99.9 % sequence similarity to that of the type strain of Herbaspirillum huttiense (Leifson 1962) Ding and Yokota 2004. This high phylogenetic relatedness of H. putei and H. huttiense was confirmed by the results of DNA-DNA hybridization between H. huttiense DSM 10281(T) and H. putei ATCC BAA-806(T) (reassociation value 96 %). Therefore, it is proposed to reclassify the type strain of H. putei as a strain of H. huttiense. However, the genome of the type strain of H. putei is about 0.9 Mb larger than that of the H. huttiense type strain. This results in a decrease in the reassociation value in the reciprocal DNA-DNA hybridization to 72 %, a level slightly above the threshold for delineating bacterial species. These data and distinctive phenotypic characteristics indicate that the name Herbaspirillum putei is a later heterotypic synonym of Herbaspirillum huttiense and permit the description of two novel subspecies, Herbaspirillum huttiense subsp. huttiense subsp. nov. (type strain ATCC 14670(T) =JCM 21423(T) =DSM 10281(T)) and Herbaspirillum huttiense subsp. putei subsp. nov., comb. nov. (type strain 7-2(T) =JCM 21495(T) =ATCC BAA-806(T)). Three bacterial strains, IEH 4430(T), IEH 4515 and IEH 8757, isolated from water were found to be the closest relatives of these strains. Strain IEH 8757 was classified as a strain of H. huttiense subsp. putei. Studies of genotypic and phenotypic features of strains IEH 4430(T) and IEH 4515 showed that the strains represent a novel species, which is most closely related to H. huttiense and for which the name Herbaspirillum aquaticum sp. nov. is proposed (type strain IEH 4430(T) =DSM 21191(T) =ATCC BAA-1628(T)).