OBJECTIVES: THE MAIN PURPOSE OF CURRENT STUDY IS ISOLATION THE YEASTS WHICH ARE CAPABLE OF PRODUCING RIBOFLAVIN.METHODS: YEASTS WERE ISOLATED FROM DIFFERENT SAMPLES OF SOIL. FIRST OF ALL, SAMPLES WERE SUSPENDED IN DISTILLED WATER AND YM BROTH MEDIUM, THEN A KNOWN AMOUNT OF THEM WERE SPREAD ON YGC AGAR. ALL COLONIES OF YEAST WERE EXAMINED FOR RIBOFLAVIN PRODUCTION. THE CULTURES OBTAINED AT THE END OF INCUBATION PERIOD WERE CENTRIFUGED AND QUANTITATIVE ESTIMATION OF VITAMIN IN THE SUPERNATANT WAS DETERMINED SPECTROPHOTOMETRIC ALLY AT 440NM. RIBOFLAVIN IDENTIFICATION WAS CARRIED OUT BY THIN LAYER CHROMATOGRAPHY AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. IDENTIFICATION OF YEAST WAS DONE BY BIOCHEMICAL AND MOLECULAR TESTS.RESULTS: IN THIS RESEARCH, 35 YEASTS WERE ISOLATED FROM SOIL OF FRUITS AND SUGARCANE. 14 ISOLATE SHOWED THE ABILITY OF RIBOFLAVIN PRODUCTION BY SPECTROPHOTOMETER THAT ACCORDING TO THE CHROMATOGRAPHICAL ANALYSIS, 10 ISOLATE OF THEM DETECTED AS FINAL RIBOFLAVIN PRODUCER. ALL OF THE PRODUCER YEASTS WERE IDENTIFIED BY BIOCHEMICAL METHODS AND MOLECULAR IDENTIFICATION WAS DONE FOR BEST PRODUCER YEAST.DISCUSSION: YEASTS ARE A GROUP OF UNICELLULAR MICROORGANISMS MOST OF WHICH BELONG TO THE FUNGI DIVISION OF ASCOMYCOTA AND FUNGI IMPERFECTI. TODAY YEASTS ARE USED AS ALTERNATIVE SOURCES OF ENZYMES, VITAMINS AND HAVE NUMEROUS APPLICATIONS IN THE HEALTH FOOD INDUSTRY AS FOOD ADDITIVES AND SO ON. RECENTLY THE RIBOFLAVIN PRODUCTION IS SHIFTING FROM CHEMICAL SYNTHESIS TO MICROBIAL PRODUCTION DUE TO ADVANCES IN METABOLIC AND GENETIC ENGINEERING. IN PRESENT STUDY A SIMPLE METHOD WAS USED TO ISOLATE YEASTS CAN PRODUCE VITAMIN.