Three Gram-negative, rod-shaped, non-spore-forming bacteria (strains CCUG 52769T, CCUG 52770 and CCUG 52771) isolated from haemodialysis water were characterized taxonomically, together with five strains isolated from industrial waters (CCUG 52428, CCUG 52507, CCUG 52575T, CCUG 52590 and CCUG 52631). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these isolates belonged to the class Betaproteobacteria and were related to the genus Pelomonas, with 16S rRNA gene sequence similarities higher than 99% with the only species of the genus, Pelomonas saccharophila and to Pseudomonas sp. DSM 2583. The type strains of Mitsuaria chitosanitabida and Roseateles depolymerans were their closest neighbours (97.9 and 97.3% 16S rRNA gene sequence similarity, respectively). Phylogenetic analysis was also performed for the internally transcribed spacer region and for three genes [hoxG (hydrogenase), cbbL/cbbM (Rubisco) and nifH (nitrogenase)] relevant for the metabolism of the genus Pelomonas. DNA-DNA hybridization, major fatty acid composition and phenotypical analyses were carried out, which included the type strain of Pelomonas saccharophila obtained from different culture collections (ATCC 15946T, CCUG 32988T, DSM 654T, IAM 14368T and LMG 2256T), as well as M. chitosanitabida IAM 14711T and R. depolymerans CCUG 52219T. Results of DNA-DNA hybridization, physiological and biochemical tests supported the conclusion that strains CCUG 52769, CCUG 52770 and CCUG 52771 represent a homogeneous phylogenetic and genomic group, including strain DSM 2583, clearly differentiated from the industrial water isolates and from the Pelomonas saccharophila type strain. On the basis of phenotypic and genotypic characteristics, these strains belong to two novel species within the genus Pelomonas, for which the names Pelomonas puraquae sp. nov. and Pelomonas aquatica sp. nov. are proposed. The type strains of Pelomonas puraquae sp. nov. and Pelomonas aquatica sp. nov. are CCUG 52769T (=CECT 7234T) and CCUG 52575T (=CECT 7233T), respectively.