Abstract: A strain of Citrobacter was isolated from the hypersaline Dead Sea of Jordan. The strain (DS1) was identified by molecular and biochemical methods. Based on 16S rRNA gene analysis, the isolate is most related to Citrobacter freundii and Citrobacter murliniae (maximum similarity is 94%). Relative to other citrobacters, the strain has a relatively high GC content (55%). The biochemical tests showed that the strain is Gram-negative, oxidase-negative and ferment lactose as other enteric bacteria. DS1 utilizes citrate as sole carbon source as other citrobacters. However, the main differences between our strain and other citrobacters is that the isolate is mild halophilic (optimum NaCl concentration in the growth medium is 4%) and o-nitrophenyl-β-galactosidase (ONPG)-negative. Based on the differences in the physiological and biochemical properties, as well as low 16S rRNA sequence similarity of this novel isolate, DS1 is suggested as new species of Citrobacter in the family Enterobacteriaceae.