Strain AM-8B5(T), isolated from Lake Sevan in Armenia, was characterized phenotypically, chemotaxonomically and phylogenetically. This chemo-organoheterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain grew on NSY medium at NaCl concentrations of 0.0-0.2 % (w/v) and at 4-30 °C. Whole-cell fatty acids were dominated by summed feature 3 (including C(16 : 1)ω7c and iso-C(15 : 0) 2-OH), C(16 : 0) and C(18 : 1)ω7c. C(12 : 0) 2-OH and C(16 : 1) 2-OH were the only hydroxylated fatty acids detected. Phylogenetic analysis as well as phenotypic and chemotaxonomic similarities indicated that the novel isolate was affiliated with the genus Polynucleobacter. 16S rRNA gene similarity values with the four previously described Polynucleobacter species ranged from 96.2 to 98.7 %. DNA-DNA hybridization experiments showed that the isolate did not belong to any of the previously described Polynucleobacter species. The isolate could be distinguished from all previously established Polynucleobacter species based on chemotaxonomic and phenotypic traits. The bacterium possessed a free-living lifestyle and represents a group of bacteria inhabiting the water column of many freshwater lakes. Based on the revealed phylogeny, and chemotaxonomic and phenotypic differences to previously described Polynucleobacter species, it is proposed that the isolate represents a novel species, Polynucleobacter difficilis sp. nov.; the type strain is AM-8B5(T) ( = DSM 22349(T) = CIP 110078(T)).