Pupal age is critical when sterilizing fruit fly pupae for field releases in the sterile insect technique (SIT) programme. When kept at 26 °C, pupae of the West Indian fruit fly, Anastrepha obliqua (Maquart), are irradiated after 12–13 d or 22 d before emergence. At this age, pupal eye color, which is used to determine the optimum stage, is very dark brown and grayish green as classified based on the Munsell® Soil Color Charts. However, it is often necessary to use different pupal holding temperatures in order to manipulate pupal development, especially when unforeseen problems occur during the rearing procedure for A. obliqua. Holding pupae at lower temperatures delays pupal development and slows down the progression of eye color changes, but at higher temperatures, the opposite occurs. The pupal eye color of the fruit fly was documented at different ages at different holding temperatures. Using this eye color as the reference guide for timing the irradiation of pupae, the optimum pupal age for irradiation when held at 15, 19, 28°C and natural environment (24–34°C) was 35–39, 28–30, 11 and 12–13 d old, respectively. The results indicate that for the A. obliqua used for SIT programs anywhere in the world, pupae destined for radiation sterilization can be maintained at holding temperatures between 15°C and 28°C without affecting their development. Pupal eye colors identified in each holding temperature can be used as baseline information in the mass rearing facility to judge the optimum time for radiation sterilization of pupae kept at the required holding temperature to accelerate or delay pupal development. Pupal eye color is a very useful tool to avoid or solve potential problems that may be encountered in mass rearing operations in the SIT release program.