For the production of riboflavin, strain development of Ashbya gossypii NRRL Y-1056 was attempted by NTG(N-methyl-N`-nitro-N-nitrosoguanidine) treatment. The optimum composition of culture medium and other culture conditions for the production of riboflavin by selected mutant Ashbya gossypii JAG-13 were determined. The optimum composition of medium was 9% of corn oil, 3% of gellatone, 4% of CSL, 0.3% of glycine, 0.2% of S770. The optimum culture temperature and initial pH of medium was 28℃ and 6.5, respectively. oxygen was essential for the production of riboflavin, but excess oxygen inhibit the production of riboflavin. When Ashbya gossypii JAG-13 was cultured under above conditions for 12 days with a bioreactor, 6.9 ㎎/㎖ of riboflavin was produced.