Spray analysis is used to characterize the fuel spray evolution and spray shape, which affects in-cylinder combustion and particulate emission characteristics of compression ignition (CI) engines. In this study, spray evolution of biodiesel blends and mineral diesel was captured using a high-speed charge coupled device (CCD) camera at different fuel injection pressures (FIPs) and ambient pressures (APs) in a constant volume spray chamber (CVSC). Results showed that spray parameters were significantly affected by FIP and AP. Higher FIPs resulted in longer fuel spray penetration length (Ls) and reduced spray cone angle (As). However, AP variation showed an exactly opposite trend of Ls and As. Increasing AP resulted in shorter Ls and increased As. Fuel properties also affected the spray characteristics, which slightly improved for lower biodiesel blends (B20: 20% v/v blend of biodiesel with mineral diesel) and then degraded for higher biodiesel blends (B40: 40% v/v blend of biodiesel with mineral diesel) with respect to baseline mineral diesel. The effects of these findings of fuel spray analysis were validated using engine experiments, which were performed in a single-cylinder research engine using identical test fuels and fuel injection parameters. Relatively superior combustion of B20-fueled engine and lower particulate emissions at higher FIPs showed good agreement with spray results.