Increasing costs of crude oil act as a driving force for developing novel methods to produce biofuels from feedstocks. The genetic engineering of the plants that provide stock for biofuels will also be required to make biofuels viable and cost-effective. The fourth-generation biofuels hold the key for renewable energy from feedstocks that do not compete with the food chain. Bioenergy production by cellulose hydrolysis and microbial synthesis calls for unique optimization of sugars and metabolic intermediates. Today, plant biologists and microbiologists are creating transgenic plants with changed, energy storing cell walls and changed reproductive organs. This type of functional reorganization for directing carbon flow through specific pathways or for directly constructing the final bioenergy compounds is the focus for this new generation of dedicated bioenergy crops. In the present chapter, an attempt was made to overview the fourth-generation biofuels under the following lines: (1) to enlist key characteristics of fourth-generation biofuels, (2) to discuss the genetic engineering techniques for biofuel production, (3) to highlight the biofuel production from genetically engineered microorganisms, (4) to focus on lignocellulosic biofuel enhancement through genetic engineering, (5) to overview the genetic engineering for lipid-based biofuels, (6) to discuss the potential role of synthetic biology and artificial pathways for fourth-generation biofuel production, and (7) to suggest future prospective of fourth-generation biofuels.