Ovarian dissection of Glossina austeni Newstead, G. brevipalpis Newstead, G. morsitans morsitans Westwood and G. pallidipes Austen shows that there are significant interspecific differences in egg-follicle lengths. Differences between G. brevipalpis and G. pallidipes are slight as are those between G. austeni and G. m. morsitans, but the former two species have significantly larger eggs at all ages examined than the latter two. Egg-follicle length is positively related to species size (measured as wing vein length) but only weakly related to the size of individuals within a species. In the ovarian dissection technique for ageing tsetse flies, nulliparous females are usually divided into 0A (one to four days) and OB (five to eight days) categories, depending on whether the first egg follicle is less or more than 0.6 mm in length. The results presented, however, show that at four days the first egg follicle is about 1 mm in length in G. pallidipes and G. brevipalpis and 0.77 mm in length in G. m. morsitans and G. austeni. Thus, application of the 0.6 mm, four-day threshold will result in severe under-estimation of the proportion of females of the former two species of less than five days of age in a population.