Biomass is an important energy resource for producing bioenergy and growing the global economy whilst minimising greenhouse gas emissions. Many countries, like Australia have a huge amount of biomass with the potential for bioenergy, but non-edible feedstock resources are significantly under-exploited. Hence it is essential to map the availability of these feedstocks to identify the most appropriate bioenergy solution for each region and develop supply chains for biorefineries. Using Australia as a case study,we present the spatial availability and opportunities for second and third generation feedstocks. Considerations included current land use, the presence of existing biomass industries and climatic conditions. Detailed information on the regional availability of biomass was collected from government statistics, technical reports and energy assessments as well as from academic literature. Second generation biofuels have the largest opportunity in New South Wales, Queensland and Victoria (NSW, QLD and VIC) and the regions with the highest potential for microalgae are Western Australia and Northern Territory (WA, NT), based on land use opportunity cost and climate. The approach can be used in other countries with a similar climate. More research is needed to overcome key technical and economic hurdles.