The present study optically measures the liquid spray penetration using high-speed backlight illumination imaging in a running small-bore compression-ignition engine. This imaging technique utilizes high-power LED as a light source that is reflected on the flat cylinder head surface except the vaporizing spray region. The boundary detection of this dark region is performed to calculate the spray tip penetration. The liquid spray development was visualized for 3 custom-made fuels exhibiting identical physical properties except the cetane number (CN30, CN40, and CN50) and a range of the distillation curves. Because the applicable injection timing range is more advanced for a lower cetane number fuel and vice versa, it provides an ample opportunity to discuss the effects of varying ambient gas temperature/density on the spray. For all tested conditions, the high-speed backlight illumination imaging was repeated for 30 injections. The results showed similar initial increase of the spray penetration for all tested injection timings and fuels due to the strong injection momentum. However, the later spray penetration showed a measurable variation with the maximum penetration becoming longer for both earlier and later injections off from TDC. The trends indicate increased spray penetration due to decreased mixing-limited vaporization at lower ambient gas temperature/density conditions. This was further supported by longer tip penetration for a fuel with higher distillation temperatures. The trends were successfully predicted using a transient jet mixing model employing discrete control volumes, suggesting indeed mixing-limited vaporization governs the liquid spray penetration in a small-bore engine.