Life-cycle assessment (LCA) has been applied to many biofuel and bioenergy systems to determine potential environmental impacts, but the conclusions have varied. Different methodologies and processes for conducting LCA of biofuels make the results difficult to compare, in-turn making it difficult to make the best possible and informed decision. Of particular importance are the wide variability in country-specific conditions, modeling assumptions, data quality, chosen impact categories and indicators, scale of production, system boundaries, and co-product allocation. This study has a double purpose: conducting a critical evaluation comparing environmental LCA of biofuels from several conversion pathways and in several countries in the Pan American region using both qualitative and quantitative analyses, and making recommendations for harmonization with respect to biofuel LCA study features, such as study assumptions, inventory data, impact indicators, and reporting practices. The environmental management implications are discussed within the context of different national and international regulatory environments using a case study. The results from this study highlight LCA methodology choices that cause high variability in results and limit comparability among different studies, even among the same biofuel pathway, and recommendations are provided for improvement.