A moderately acidophilic, facultative chemoautotrophic, As(III)-oxidizing Thiomonas sp. (strain 3As(T)) was previously shown, on the basis of comparative 16S rRNA gene sequences, to be closely related to both Tm. perometabolis DSM 18570(T) and Tm. intermedia DSM 18155(T). While it had shared many physiological traits with Tm. intermedia (T), a mean DNA-DNA hybridization value (DDHV) of 47.2% confirmed that strain 3As(T) was not a strain of Tm. intermedia, though the situation with regard to Tm. perometabolis (DDHV previously determined as 72%) was more ambiguous. A comparative physiological and chemotaxonomic study of strain 3As(T) and Tm. perometabolis (T) was therefore carried out, together with multilocus sequence analysis (MLSA) of all three bacteria. Differences in fatty acid profiles and utilization of organic substrates supported the view that strain 3As(T) and Tm. perometabolis are distinct species, while MLSA showed a closer relationship between strain 3As(T) and Tm. intermedia (T) than between strain 3As(T) and Tm. perometabolis (T). These apparent contradictory conclusions were explained by differences in genome of these three bacteria, which are known to be highly flexible in Thiomonas spp. A novel species designation Thiomonas arsenitoxydans is proposed for strain 3As(T) (DSM 22701(T), CIP 110005(T)), which is nominated as the type strain of this species.