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A Lactobacillus strain, IMCC1736T, was isolated recently from a Korean freshwater pond following an extensive study of the microbial community in this ecosystem. Its 16S rRNA gene was sequenced and phylogenetic analysis placed this strain within the Lactobacillus salivarius group, closely related to Lactobacillus satsumensis NRIC 0604T, with 97.9 % sequence similarity. In the present work, the taxonomic status of strain IMCC1736T has been re-evaluated. It was characterized phylogenetically, genotypically and phenotypically and, based on DNA–DNA hybridization values, this strain represents a novel Lactobacillus species. Strain IMCC1736T can be differentiated genotypically from its closest relatives by randomly amplified polymorphic DNA analysis and ribotyping patterns; phenotypically, it can be distinguished by its inability to grow in 5 % NaCl and at pH 3.3 and by certain carbohydrate fermentations. Strain IMCC1736T is Gram-positive, catalase-negative and microaerophilic. Cells are motile rods and show homofermentative metabolism. The name Lactobacillus aquaticus sp. nov. is proposed, with strain IMCC1736T (5CECT 7355T 5DSM 21051T) as the type strain. The genus Lactobacillus belongs to the lactic acid bacteria group and is a highly heterogeneous genus in terms of th