A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, aerobic bacterial strain, designated 16F7C-2(T), was isolated from the Han River, South Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 16F7C-2(T) belonged to the family Cytophagaceae in the phylum Bacteroidetes and was most closely related to Hymenobacter algoricola VUG-A23a(T) (98.3%) and Hymenobacter fastidiosus VUG-A124(T) (97.7%). The G + C content of the genomic DNA of strain 16F7C-2(T) was 63.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone; a fatty acid profile with summed feature 3 (C-16:1 omega 7c/C-16:1 omega 6c; 19.5%), summed feature 4 (C-17:1 iso I/C-17:1 anteiso B; 15.9%), C-15:0 iso (12.6%), C-16:1 omega 5c (10.5%), and C-15:0 anteiso (10.4%) as the major components; and a polar lipid profile with phosphatidylethanolamine as the major component also supported the affiliation of strain 16F7C-2(T) to the genus Hymenobacter. The DNA-DNA relatedness between strain 16F7C-2(T) and H. algoricola JCM 17214(T) and H. fastidiosus JCM17224(T) were 45.2 +/- 5.8 and 40.3 +/- 2.9%, respectively, clearly showing that the isolate constitutes a new genospecies. Strain 16F7C-2(T) could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic, and chemotaxonomic features. Therefore, strain 16F7C-2(T) represents a novel species of the genus Hymenobacter, for which the name Hymenobacter knuensis sp. nov. is proposed. The type strain is 16F7C-2(T) (=KCTC 52538(T) = JCM 31814(T)).