Microorganisms are the key component for conversion of different raw materi­als into ethyl alcohol during the fermentation step. The success of the overall fuel ethanol production process depends on the selected microbial strains. In this chapter, some metabolic features of microorganisms used for ethanol biosynthe­sis are discussed. The development of efficient, low-cost, and environmentally friendly processes for fuel ethanol production requires the selection of suitable microorganisms that contribute to achieving such goals. In addition, the devel­opment of microbial strains allowing innovative designs and making use of the wide availability of feedstock resources, especially lignocellulosic materials, is required. For this reason, the main strategies for genetic modification of microor­ganisms with better performance during ethanol production from different feed­stocks are discussed in this chapter.