Among the many types of alternative fuels, biodiesel has emerged as an eco-friendly fuel for the compression-ignition engines. Investigations into biodiesel combustion have been performed with various aspects and methodologies. In this study, the effects of the pilot injection strategies on the flame temperature and soot distribution were investigated experimentally and numerically using an optical CI engine fueled with biodiesel. The endoscopy system captured the spray development and combustion processes in the cylinder. In addition, obtained pictures were used to estimate the flame temperature and soot distribution using a post-processing program. Furthermore, the KIVA/CHEMKIN code was used to enhance the analysis of the pilot combustion characteristics. These numerical results allowed information that cannot be obtained from physical experiments. The experimental conditions were composed of various pilot injection timings and quantities. In the present experiment, the experimental conditions were fixed with the exception of the injection strategy that was used. The experimental results showed that the flame temperature decreased as the pilot fuel mass increased. Additionally, it was also found that biodiesel combustion generated a higher flame temperature and lower soot concentrations compared to neat diesel combustion.