This study examines the effects of biodiesel carbon chain length on the development of high-temperature flame and soot in single-cylinder, light-duty, optical diesel engine. Planar laser-induced fluorescence of OH (OH-PLIF) and laser-induced incandescence (soot-PLII) are performed separately using petroleum diesel and two biodiesel surrogate fuels. The selected surrogate fuels have a saponification number (SN) of 330 and 233 for shorter and longer carbon chain length, respectively, while the iodine value (IV) characterising the degree of unsaturation is kept similar. The laser-based images are shown together with the chemiluminescence images of naturally occurring cool-flame signals and electronically excited OH (OH*). The start of high-temperature reaction timing was matched for the tested fuels by adjusting the injection timing so that the in-cylinder ambient conditions at the start of combustion were consistent. The results show that the longer carbon chain biodiesel has shorter ignition delay time, which makes a significant impact on the spatial and temporal development of high-temperature reaction zones. The OH signals for the longer carbon chain length biodiesel are observed close to the piston-bowl wall whereas they appear later in the penetrating front of the wall-interacting jet for the shorter carbon chain length biodiesel. Both fuels show soot formation occurring in the wall-jet head region while the longer carbon chain length biodiesel has higher soot concentration and larger area than that of the shorter carbon chain biodiesel. This is due to lower pre-combustion mixing, reduced fuel oxygen content, and possibly less significant OH-induced soot oxidation.