High fructose corn syrup (HFCS) 55 (HFCS55) is a sweetener made from corn composed of 55% fructose and a mix of glucose and minor amounts of short chain oligosaccharides. It is widely used in food applications as a sweetener, preservative, flavor enhancer, moisture retainer, and to provide texture. The objective of this study was to assess the cradle-to-gate environmental impacts of average U.S. production of HFCS55. Our assessment was based on confidential primary data supplied by 13 participating facilities, including material and energy inputs and emissions for milling, refining and coproduct drying. We estimated life cycle impacts using the Tool for the Reduction and Assessment of Chemical and Environmental Impacts (TRACI 2.1). Additional impact categories were included for land use (midpoint H), Global Warming Potential (100 years) (GWP), and Cumulative Energy Demand. Environmental hotspots identified in the production of HFCS55 indicated that direct emissions from the conversion process contribute over 50% of the global warming potential, as well as the overwhelming bulk of energy and fuels used in the system, 35%–40% of acidification and ecotoxicity impact potentials and potential respiratory effects caused by particulate matter. Coal and natural gas extraction and combustion accounted for 70%–90% of the global warming impacts for process heat and power, and their extraction also contribute significantly to acidification and ecotoxicity. About 40% of the global warming potential for HFCS55 was generated by fuel used in processing for the aggregated industry average HFCS55. Nearly half of the HFCS55 was produced in facilities that utilize coal in cogeneration of steam and electricity, and that grouping had almost 20% higher impact than the U.S. aggregated. Replacing coal in the industry average with natural gas could lower impact about 20%; for the coal-using cogeneration group, that benefit would be larger. Replacing all coal and natural gas with biogas from process residues potentially could more than halve the global warming potential depending on which grouping a facility was in, although this scenario needs further evaluation. A sensitivity analysis of energy replacement options for HFCS55 showed that transition from coal to natural gas or other energy sources would lower the GWP and other impacts. This assessment was the first study of its kind; further work is needed to explicitly define areas for industry improvement. This study focuses primarily on High Fructose Corn Syrup (HFCS), specifically HFCS55, production. This work addresses that crucial gap by providing the most up-to-date, industry-relevant life cycle assessment (LCA) and associated life cycle inventory available for corn wet milling and HFCS55 and other products. It also provides a life cycle inventory for the production of U.S. corn used for wet milling and refining. Prior to this work, there are no published analyses of the life cycle environmental impacts of HFCS 55 (or associated sweeteners) reflecting industry practice from primary data at industry scale. Indeed, there are very few assessments of the environmental impact of HFCS as a product, industry-scale or otherwise.