Biofuels have been identified as a potential short-term solution for reducing greenhouse gas (GHG) emissions from road transport. In order to ensure that they successfully deliver emission savings, the overall GHG balance of producing them must be calculated accurately, and compared with conventional fossil fuels. Life cycle assessment has dominated the process of assessing the GHG emissions from biofuels, though the results can vary significantly, due to real variation caused by biomass feedstock types, processing stages, and uncertainty in GHG emission from certain processes, but also due to how the GHG emission balance is calculated. This study has examined the relative importance of ‘scientific’ variation, and that caused by different methodological approaches. Three case studies with different methodological issues associated with accounting for their GHG emissions were developed. The variation in the LCA results due to the variability of inputs and outputs and from uncertainty of emissions from certain processes were assessed. The results showed that there is a high amount of variation in GHG emissions from fertiliser use, nitrous oxide emissions from soil and direct land use change. The GHG emissions from the full bioethanol production system were then calculated according to three specific GHG regulatory methodologies that are either currently used, or have been used in the UK for biofuels, products and services. This study has found that LCA methodology can cause considerable variation in differences in LCA results. The variation is sometimes caused by arbitrary decisions concerning how GHG emissions from a process should be accounted for and attributed to the main product. Variation in the results that is caused by methodology is comparable to that caused by scientific variation, which is ‘real’ that sometimes cannot be avoided without detailed study. The different results are due to the approach the methodology takes to LCA; whether the method tends toward attributional or consequential LCA. For reporting purposes, the European Directive’s Renewable Energy Directive (RED) states that attributional LCA (ALCA) is best as it provides a snapshot of emissions that are released, and attributable to the production and use of the product or service. A consequential LCA (CLCA), on the other hand is better suited for policy analysis as the potential impacts are applicable to a wider, even global scope. None of the methodologies studied completely adheres to ACLA or CLCA. It appears that they have confused the two within their calculation rules; therefore they do not fulfil their goal and scope.A critical assessment of the accounting methodology within RED is also performed here. The results show that the methodology is inconsistent and arbitrary, and currently too vague to be practical for GHG reporting. The results from this study indicate that the RED penalises the use of renewable energy and its calculation methodology does not support 2nd generation bioethanol production.