The common realization of the necessity to reduce the use of mineral sources is promoting the use of alternative fuels. Big efforts are being made to replace petroleum derivatives in the internal combustion engines (ICEs). For this purpose it is mandatory to evaluate the behavior of non-conventional fuels in the ICEs.The optical diagnostics have proven to be a powerful tool to analyze the processes that take place inside the engine. In particular, 2d imaging in the infrared range can reveal new details about the effect of the fuel properties since this technique is still not very common.In this work, a comparison between commercial diesel fuel and two non-conventional fuels has been made in an optically accessible diesel engine. The non-conventional fuels are: the first generation biofuel Rapeseed Methyl Ester (RME) and an experimental blend of diesel and a fuel with high glycerol content (HG). Both a CCD visible camera and an infrared camera have been used to record images from the combustion chamber. The infrared range investigated is from 1.5 to 5 μm; moreover, a band pass filter at 4.2 μm has been used to impress the radiation of the CO2 molecules while a filter at 3.9 μm has been used to visualize the fuel location in terms of HCs, and the reacting spray by intermediate products of the combustion process. The engine mounts the head of a real Euro5 diesel engine and a Common Rail injection system with solenoid injector. The engine head is placed on an elongated piston and an optical access in the piston provides the bottom view of the combustion chamber. Images are taken by means of a 45° IR-mirror located in the elongated piston. The engine runs in two operating points of the New European Driving Cycle in order to investigate the mixing process in conditions similar to those of the real engine.The infrared imaging has proven to give important information about the in-cylinder reactions. The pilot injection vaporization and mixing has been evaluated. The interaction between the main injection and the reacting fuel has been analyzed. Finally, CO2 plumes appear at the end of main injection and they are isolated on the tip of the jets and close to the chamber wall.