The connection between energy use and water scarcity is not well understood. The production of energy requires water and the supply of water requires energy. Water already plays a major role in stationary energy production; thermoelectric power generation is responsible for nearly half of total freshwater withdrawals in the United States. Current transportation fuels, which account for approximately one-third of U.S. energy consumption, are not nearly as reliant on freshwater given that petroleum fuel production makes up just a few percent of U.S. water use. If transportation were to become more reliant on water-intensive sectors such as power generation and agriculture, there would be major implications for water availability in the United States. As electricity and biofuels gain a larger share of the market, this is exactly the transition that is taking place. Inconsistent water use metrics, inappropriate impact allocation practices, limited system boundaries due to lack the necessary tools and data, and the failure to quantify water resource availability and greenhouse gas (GHG) impacts are common pitfalls of existing assessments of transportation energy-related water use. To fill the knowledge gaps, this dissertation proposes a comprehensive life-cycle framework for assessing the water withdrawals and consumption of current and near-future U.S. transportation fuels -- including gasoline, bio-based ethanol, and electricity. With this proposed framework for performing a life-cycle inventory and impact assessment, the following three questions are answered:1. What is the life-cycle water footprint of current and near-future transportation fuel production in the United States?2. How might U.S. transportation fuel production pathways impact freshwater availability in the future?3. What is the greenhouse gas-intensity of the water required for transportation fuel production, and how do these emissions impact the overall transportation fuel greenhouse gas footprints?Understanding the impacts of water use on freshwater resources and GHG emissions requires knowledge of not only the fuel production pathways, but also how these pathways interact with other sectors in the economy. As new transportation fuels emerge, demand for some goods and services will increase while for others it will decrease, and each change has an effect on overall water demand. Quantifying the net system-wide impact of producing these new fuels is key to understanding the water implications of transportation energy-related policy decisions. Furthermore, by geospatially disaggregating predicted water requirements for transportation fuel production pathways at the U.S. county-level, locations within the United States can be identified as vulnerable to local surface and groundwater shortages. These shortages may result in high water prices and the need for energy-intensive water supply methods such as desalination, importation, or wastewater recycling. Identifying regions with vulnerable water resources allows decision makers in industry and the public sector to guide burgeoning transportation fuel markets in ways that maximize their contributions to energy independence and greenhouse gas emissions reductions while avoiding negative impacts on water availability. Results from the U.S. analysis show that indirect water use has a significant impact on total water use, particularly for withdrawals. In no other pathway is this as pronounced as it is for cellulosic ethanol production (in this case, corn stover and Miscanthus to ethanol). By using system expansion to account for the electricity generation displaced by cellulosic biorefineries' exports to the grid, total water consumption for those pathways drops considerably and total withdrawals actually becomes a net negative number. When the inventory is geospatially disaggregated and compared to drought and groundwater vulnerability data, the results show that biofuel production concentrated in the Midwest puts pressure on the already-overpumped High Plains Aquifer. Petroleum fuel production pathways result in water use concentrated in locations that are predicted to experience long-term drought, specifically California, Texas, and Wyoming. Electricity, in contrast, is more widely distributed throughout the U.S., but the high surface water consumption rates in the western half of the country may exacerbate future surface water shortages in those regions. Gaining a better knowledge of how the production and consumption of fuels impacts freshwater resources is absolutely critical as humans attempt to transition into a more sustainable energy future. By making contributions to the methodologies required to assess the environmental impacts of water use, as well as knowledge about the potential water impacts of current and near-future U.S. transportation fuels, this dissertation provides U.S. decision makers with information necessary to create the most economical and sustainable transportation energy future possible while also providing future researchers with the tools to answer questions that have yet to be asked.