In the last decade, fuels from renewable resources have become increasingly more interesting with regard to transport applications because of their biodegradability, potential improvements on exhaust emission, and benefits regarding a virtuous CO2 cycle of the earth. Within this framework fuels produced in South Africa and Europe have been analyzed, each being different in nature. The injection process and air-fuel mixture preparation of soybean methyl ester (SME), rapeseed methyl ester (RME), and gas to liquid (GTL) were compared with commercial diesel fuel. An experimental investigation of the fuel spray under both nonevaporative and evaporative conditions is reported. Measurements were carried out in two optically accessible systems: a high-pressure vessel at ambient temperature and a single-cylinder diesel engine for the evaporative conditions. The two systems are equipped with a Bosch second-generation common rail solenoid-driven fuel injection system with a seven-hole nozzle. Double injection strategies (pilot and main), corresponding to the operating conditions of the commercial Euro 5 diesel engine, have been obtained via fully opened electronic control units. The characteristics of the investigated sprays include the fuel delivery, the instantaneous flow rate, jet visualization, the liquid penetration, and break-up individuation. Different spatial distributions of the liquid fuel in the chamber have been determined. Moreover, comparisons of the experimental penetration data with those of Hiroyasu and Siebers theoretical correlation have been made. Finally, a new experimental approach for determination of the break-up length based on the image luminosity distribution analysis has been proposed.