Potential global biodiversity impacts from near-term gasoline production are compared to biofuel, a renewable liquid transportation fuel expected to substitute for gasoline in the near term (i.e., from now until c. 2030). Petroleum exploration activities are projected to extend across more than 5.8 billion ha of land and ocean worldwide (of which 3.1 billion is on land), much of which is in remote, fragile terrestrial ecosystems or off-shore oil fields that would remain relatively undisturbed if not for interest in fossil fuel production. Future biomass production for biofuels is projected to fall within 2.0 billion ha of land, most of which is located in areas already impacted by human activities. A comparison of likely fuel-source areas to the geospatial distribution of species reveals that both energy sources overlap with areas with high species richness and large numbers of threatened species. At the global scale, future petroleum production areas intersect more than double the area and higher total number of threatened species than future biofuel production. Energy options should be developed to optimize provisioning of ecosystem services while minimizing negative effects, which requires information about potential impacts on critical resources. Furthermore, energy conservation and identifying and effectively protecting habitats with high-conservation value are critical first steps toward protecting biodiversity under any fuel production scenario.