Betaine is a trimethylglycine, serves as osmoregulator to prevent dehydration and plasmolysis under adverse hyperosmotic environments. Choline oxidase gene from Arthrobacter sp. catalyzes two step oxidation reaction of choline to betaine followed by betaine accumulation in cells which in turn help them to survive and thrive in harsh environmental condition. To identify potential choline oxidase gene source, a gram stain positive, rod shaped, catalase and oxidase positive, motile, aerobic bacterial strains designated as HYJE003 and HYJE005 was isolated based on the colony morphology, biochemical and molecular characterization from the industrially polluted soil samples of Hyderabad, India. Optimum growth of the isolated strains was observed at 32°C on nutrient agar media and was found that both the strains were capable of utilizing variety of sugars as carbon source. The 16S rRNA gene sequence analysis revealed that strain HYJE003 was closely related to Arthrobacter globiformis with pairwise sequence similarities of 99.85%, 99.63%, 98.76% and 98.12% respectively. The strain HYJE005 was closely related to Arthrobacter phenanthrenivorans with pairwise sequence similarities of 99.93%, 99.47%, 99.25% and 98.11% respectively. Choline oxidase gene potential of the isolates was studied by feeding the cultures with choline chloride and converted betaine was assessed by the formation of betaine reineckate. Findings revealed that the isolated strain Arthrobacter globiformis-HYJE003 has four times higher conversion rate of choline chloride to betaine than the strain Arthrobacter phenanthrenivorans HYJE005.