Due to the rising energy demand of our modern society and the finite amounts of petroleum-based fuels, renewable forms of energy have become extremely important. Bio-based fuels like bioethanol and biodiesel represent an already applied and accepted alternative. Biodiesel is currently mainly produced from plant oils. A new and promising alternative, which avoids the use of food crops, is the cultivation of the oleaginous yeast Yarrowia lipolytica, which possesses the capacity to accumulate up to 40% of its biomass in form of lipids. Moreover, this non-conventional yeast is able to metabolize a broad range of carbon-sources, presenting a sustainable alternative to reutilize a wide spectrum of waste substrates. This makes it an auspicious candidate for the generation of non-edible oils for biodiesel production. In this work, we aimed to generate a Y. lipolytica mutant strain with enhanced lipid production when grown on wheat straw hydrolysate as sole carbon source. Therefore, UV mutagenesis was applied and mutants with a high-lipid content were selected by their ability to grow in the presence of the fatty acid inhibitor cerulenin. Further, growth of the mutants on wheat straw hydrolysate was evaluated. The fatty acid composition was analysed by GC-FID and the calculated total lipid content revealed an up to 33% increase compared to the wild type strain. Fermentation optimisation and the combination of various waste substrates as carbon sources are expected to further increase the total lipid yield by the Y. lipolytica mutant strain and serve as initial point for its industrial scale evaluation.