Three strains of methylotrophic Rhodocyclaceae (FAM1, RZ18-153 and RZ94) isolated from the Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids are 16:1omega7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the species FAM1, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus, M. universalis and M. thermotolerans. Like other methylotrophic Methyloversatilis spp. all three cultures possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), N-methylglutamate pathway, serine cycle (isocitrate lyase/malate synthase, Icl + variant). Like M. universalis the strains FAM1, RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans all new strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data the strains FAM1, RZ18-153 and RZ94 are placed in a new species, Methyloversatilis discipulorum (Type strain FAM1T, JCM = 30542; VKM = B-2888T).