Research into novel internal combustion engines requires consideration of the diversity in future fuels that may contain significant quantities of bio-components in an attempt to reduce CO2 emissions from vehicles and contribute to energy sustainability. However, most biofuels have different chemical and physical properties to those of typical hydrocarbons; these can lead to different mechanisms of mixture preparation and combustion. The current paper presents results from an optical study of combustion in a direct-injection spark-ignition research engine with gasoline, iso-octane, ethanol and butanol fuels injected from a centrally located multi-hole injector. Methane was also employed by injecting it into the inlet plenum of the engine to provide a benchmark case for well-mixed ‘homogeneous’ charge preparation. Crank-angle resolved flame chemiluminescence images were acquired and post-processed for a series of consecutive cycles for each fuel, in order to calculate in-cylinder rates of flame growth and motion. In-cylinder pressure traces were used for heat release analysis and for comparison with the image-processing results. All tests were performed at 1500 RPM with 0.5 bar intake plenum pressure. Stoichiometric (ϕ = 1.0) and lean (ϕ = 0.83) conditions were considered. The combustion characteristics were analysed with respect to laminar and turbulent burning velocities obtained from combustion bombs in the literature and from traditional combustion diagrams in order to bring all data into the context of current theories and allow insights by making comparisons were appropriate.