A Gram-stain-negative, rod-shaped, non-spore-forming bacterium (strain JM-1396(T)) producing a yellow pigment, was isolated from the healthy internal stem tissue of post-harvest cotton (Gossypium hirsutum, cultivar 'DES-119') grown at the Plant Breeding Unit at the E.V. Smith Research Center in Tallassee (Macon county), AL, USA. 16S rRNA gene sequence analysis of strain JM-1396(T) showed high sequence similarity values to the type strains of Novosphingobium mathurense, Novosphingobium panipatense (both 98.6%) and Novosphingobium barchaimii (98.5%); sequence similarities to all other type strains of species of the genus Novosphingobium were below 98.3%. DNA-DNA pairing experiments of the DNA of strain JM-1396(T) and N. mathurense SM117(T), N. panipatense SM16(T) and N. barchaimii DSM 25411(T) showed low relatedness values of 8% (reciprocal 7%), 24% (reciprocal 26%) and 19% (reciprocal 25%), respectively. Ubiquinone Q-10 was detected as the dominant quinone; the fatty acids C18 : 1ω7c (71.0%) and the typical 2-hydroxy fatty acid, C14 : 0 2-OH (11.7%), were detected as typical components. The polar lipid profile contained the diagnostic lipids diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine. The polyamine pattern contained the major compound spermidine and only minor amounts of other polyamines. All these data revealed that strain JM-1396(T) represents a novel species of the genus Novosphingobium. For this reason we propose the name Novosphingobium gossypii sp. nov. with the type strain JM-1396(T) ( = LMG 28605(T) = CCM 8569(T) = CIP 110884(T)).