Although the 2005 Environmental Protection Act (EPAct) was enacted to bolster the emerging biofuel industry, 52% of advanced biofuel (AB) projects ended by 2015. However, there are no complete lists of internal and external barriers that can help to explain why these projects are failing. The goal of this study was to develop a list of barriers impeding advanced biofuel projects by conducting a survey of biofuel stakeholders. Based on a literature review and previous research, a list of 23 hypothesized internal and external barriers was elaborated. A survey was conducted to have industry stakeholders provide their perception on the list of hypothesized barriers. The perceptions of industry stakeholders were analyzed by dividing the sample in three different stakeholder groups: advanced biofuel industry members, government representatives, and a third category called others that included publishers, journalists, suppliers, and other related stakeholders to the industry. In addition, nonparametric statistical techniques were used to compare the perceptions of the groups. Here, the most significant results indicated that Technology issues was considered as an internal barrier for the three groups while Funding and Renewable Fuel Standards were perceived as external barriers by the three groups too. In addition, the rating of barriers was further analyzed only by AB industry stakeholders in order to uncover more details on the perception of barriers that might be preventing the AB industry to prosper.