An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5–7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74–99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA–DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.