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Human African trypanosomiasis (HAT) and African animal trypanosomosis (AAT) are devastating diseases spread by tsetse flies (Glossina spp.), affecting humans and livestock, respectively. Current efforts to manage these diseases by eliminating the vector through the sterile insect technique (SIT) require transportation of irradiated late-stage tsetse pupae under chilling, which has been reported to reduce the biological quality of emerged flies. We therefore evaluated the impact of irradiation and transportation (including vibration and shock) on pupae at early-stage development (22 days of age) under ambient temperature and compared it to that on pupae at the late-stage development (29 days of age) under chilling, the current practice for tsetse in SIT programs. The quality of flies emerging from these transported pupae was assessed by their emergence rates, flight propensity, mating ability, insemination rates and survival rates (over ca. 100 days, and after specified shorter periods). Generally, flies emerging from the 22-day-old pupae had significantly (p < 0.05) higher values for the tested quality parameters, as compared to those emerging from 29-day-old pupae. Irradiation, transportation and the combination thereof significantly (p < 0.05) reduced all the tested quality parameters as compared with the untreated control within the 22-day-old pupae group. Further, vibration had a significant negative effect on the quality of flies, notwithstanding the age of the pupae. Irradiation and transportation of pupae at 22 days of age resulted in a higher proportion of flies of good biological quality as compared to those of 29 days of age, and hence may be considered for future SIT programs.