Two Gram-negative, oxidase-positive rods (strains BSB 9.5T and BSB 41.8T) isolated from wastewater were studied using a polyphasic approach. 16S rRNA gene sequence comparisons demonstrated that both strains cluster phylogenetically within the family Comamonadaceae: the two strains shared 99.9 % 16S rRNA gene sequence similarity and were most closely related to the type strains of Hydrogenophaga palleronii (98.5 %) and Hydrogenophaga taeniospiralis (98.0 %). The fatty acid patterns and substrate-utilization profiles displayed similarity to the those of the five Hydrogenophaga species with validly published names, although clear differentiating characteristics were also observed. The two strains showed DNA-DNA hybridization values of 51 % with respect to each other. No close similarities to any other Hydrogenophaga species were detected in hybridization experiments with the genomic DNAs. On the basis of these results, two novel Hydrogenophaga species, Hydrogenophaga defluvii sp. nov. and Hydrogenophaga atypica sp. nov. are proposed, with BSB 9.5T (=DSM 15341T=CIP 108119T) and BSB 41.8T (=DSM 15342T=CIP 108118T) as the respective type strains.