Like other EU Member States, Austria will meet the substitution target of the EU European Renewable Energy Directive for transportation almost exclusively by first generation biofuels, primarily biodiesel from oilseed rape (OSR). Genetically modified (GM) plants have been promoted as a new option for biofuel production as they promise higher yield or higher quality feedstock. We tested implications of GM OSR application for biodiesel production in Austria by means of high resolution spatially explicit simulation of 140 different coexistence scenarios within six main OSR cropping regions in Austria (2400 km2). We identified structural land use characteristics such as field size, land use diversity, land holding patterns and the proportion of the target crop as the predominant factors which influence overall production of OSR in a coexistence scenario. Assuming isolation distances of 800 m and non-GM-OSR proportions of at least 10% resulted in a loss of area for cultivation of OSR in all study areas ranging from -4.5% to more than -25%, depending on the percentage of GM farmers and on the region. We could show that particularly the current primary OSR cropping regions are largely unsuitable for coexistence and would suffer from a net loss of OSR area even at isolation distances of 400 or 800 m. Coexistence constraints associated with application of GM OSR are likely to offset possible GM gains by substantially reducing farmland for OSR cultivation, thus contradicting the political aim to increase domestic OSR area to meet the combined demands of food, feed and biofuel production.