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It is well known that direct injection (DI) is a technology enabler for stratified combustion in spark-ignition (SI) engines. At full load or wide-open throttle (WOT), partial charge stratification can suppress knock, enabling greater spark advance and increased torque. Such split-injection or double-pulse injection strategies are employed when using gasoline in DI (GDI). However, as the use of biofuels is set to increase, is this mode still beneficial? In the current study, the authors attempt to answer this question using two gasoline-alternative biofuels: firstly, ethanol; the widely used gasoline-alternative biofuel and secondly, 2,5-dimethylfuran (DMF); the new biofuel candidate. These results have been benchmarked against gasoline in a single-cylinder, spray-guided DISI research engine at WOT (λ = 1 and 1500 rpm). Firstly, single-pulse start of injection (SOI) timing sweeps were conducted with each fuel to find the highest volumetric efficiency and IMEP.