Glossina pallidipes salivary gland hyperplasia (GpSGH) syndrome caused by the salivary gland hyperplasia virus reduces the reproduction potential of tsetse flies, posing a serious threat for rearing of sufficient colonies for use of tsetse and trypanosome control using the sterile insect technique. This research was conducted in the Kaliti Tsetse Mass Rearing and Irradiation Centre in Ethiopia with the objective of studying the prevalence of GpSGH syndrome in laboratory colonies of G. pallidipes (Tororo and Arbaminch) reared for release in the implementation of the sterile insect technique and a field strain of G. pallidipes Arbaminch. Presence or absence of GpSGH was determined when pathological features of the salivary gland were revealed after dissection. The overall prevalence of GpSGH syndrome in laboratory colonies was 48.3% (747/1548) with a statistically significant (z = 17.30, p = 0.001) prevalence of 70.2% (544/775) in Arbaminch colonies and 26.26% (203/773) in Tororo colonies. The prevalence of GpSGH in laboratory flies fed according to the clean blood feeding protocol was 68.9% and 22.4% in Arbaminch and Tororo strains respectively. It was 70.5% and 27.2% respectively in laboratory colonies of Arbaminch and Tororo strains fed according to the standard membrane feeding protocol. The difference in prevalence of the disease between the two feeding protocols was not statistically significant in either Arbaminch (z = 0.361, p = 0.359) or Tororo (z = 1.22, p = 0.111) strains. The prevalence of SGH in wild G. pallidipes Arbaminch strain was 3% (15/500) and was significantly (z = 23.61, p < 0.001) lower than in the laboratory strain. The effect of age and density-related stress on the development of GpSGH was not statistically significant. The prevalence of GpSGH in the newly emerging (teneral) flies in the laboratory colonies was 66.7% and 20% in the Arbaminch and Tororo strains respectively. For all considered risk factors, the prevalence was much higher in G. pallidipes Arbaminch laboratory colonies.