Two anaerobic bacterial strains, designated SHI-1(T) and SHI-2, were isolated from chlorinated solvent-contaminated groundwater. They were found to be identical in phenotypic properties and shared high (98.5-99.8 %) pairwise 16S rRNA gene sequence similarity. Multiple 16S rRNA genes were found to be present in the isolates as well as Pelosinus fermentans DSM 17108(T) and Sporotalea propionica DSM 13327(T). Strains SHI-1(T) and SHI-2 could be differentiated from their closest phylogenetic relatives, P. fermentans DSM 17108(T) and S. propionica DSM 13327(T), on the basis of their phenotypic and phylogenetic properties. The isolates were Gram-negative, spore-forming, motile rods with peritrichous flagella. Growth occurred at 10-42 °C and pH 5.5-8.5. Fermentative growth was observed on Casamino acids, fructose, fumarate, glucose, glycerol, pyruvate and yeast extract. The major organic acids produced from glucose and glycerol fermentation were propionate and acetate. The major organic acids produced from fermentation of fumarate were propionate, acetate and succinate. The major cellular fatty acids were summed feature 4 (consisting of C(15:1)ω8c and/or C(15:2)), summed feature 8 (consisting of C(17:1)ω8c and/or C(17:2)) and C(14:0) dimethyl aldehyde. The polar lipids comprised aminophospholipids, including phosphatidylethanolamine and phosphatidylserine, and an unknown phospholipid. The genomic DNA G+C content was 39.2 mol%. We propose that strains SHI-1(T) and SHI-2 are assigned to a novel species of the genus Pelosinus, with the name Pelosinus defluvii sp. nov. (type strain SHI-1(T) = NRRL Y-59407(T) = LMG 25549(T)). The description of the genus Pelosinus is emended. We also propose the transfer of S. propionica to the genus Pelosinus as Pelosinus propionicus comb. nov. (type strain TmPN3(T) = DSM 13327(T) = ATCC BAA-626(T)), on the basis of phylogenetic, chemotaxonomic and phenotypic properties.