Tsetse colonized either for laboratory studies or for release in S. I. T. programmes are assumed to be healthy and geneticallyw similar to flies in natural populations. However, insect colonies are subjected to many of the same evolutionary forces that influence genetic changes in natural populations, i.e. drift, selection, hitch-hiking, mutations, assortative mating and immigration. The influence of these on genetic structure of tsetse fly colonies is outlined, and examples are presented from several species. There is little or no evidence for adaptation during the early phases of laboratory colonization of five species of tsetse. A model is presented indicating that with as little as a 5% fitness difference between males, some colonies have existed long enough to have undergone significant changes in the relative numbers of males having “standard” and “enhanced” fitness. Slight changes in heterozygosity of colonized flies is documented by comparing colonies and field-collected flies and by comparisons within colonies over periods of several generations or years. An example of hitch-hiking is illustrated with the closely linked genes Sr (sex ratio) and Est-X in Glossina morsitans submorsitans. A possible interaction between alleles at these loci is discussed. A summary is presented of polyacrylamide gel electrophoretic methods for monitoring 16 polymorphic loci distributed among the X chromosome and autosomes of tsetse.