Background and Objectives: Vitamin B2 (riboflavin) is a water-soluble vitamin and yellow. The aim of this study is to identify ‎strains of Bacillus isolated from soil do with the ability to produce riboflavin by PCR. Materials and Methods: Soil samples was mild randomly during 2 consecutive days the air. Sampling was performed of areas far from the sun ‎and from 3 to 10 cm soil depth. The genus Bacillus were identified to species level ‎using standard methods. Also isolates were approved identified using the PCR method. To study the ‎riboflavin production of synthetic culture media were used and HPLC techniques. Result: PCR using gene 16SrRNA test results showed that 32 strains obtained from a strain of Bacillus subtilis ‎and 10 were 2 strain of Bacillus megaterium. The isolated strains of Bacillus megaterium PTCC 1250 with ‎standard strains were positive of riboflavin production in synthetic medium and HPLC. ‎Conclusions: Bacillus subtilis bacteria living in the soil among the most important role in the production of ‎riboflavin and riboflavin-producing strains to confirm the use of molecular methods is necessary.