Bioethanol and distilled beverage industries need rapid, reliable and simple methods for yeast identification. This work proposes the use of primers that allow the clear and direct identification of three of the most important yeast species in alcoholic fermentation. When using the ScHO primer, it is possible to distinguish S. cerevisiae from S. pastorianus by the size of the bands. Moreover, a new primer was also presented (LgHO) for the identification of S. bayanus, with an amplification fragment of about 700 bp. Both primers used in this study have been tested with several other yeast species commonly found in bio- ethanol and cachaca industries, and amplification was not observed for any of them (Table 1).