Cytological maps have been prepared, and a detailed description is presented of all major markers in the polytene chromosomes of the tsetse fly Glossina morsitans morsitans, an important vector of African trypanosomiases. Only the five eu-chromosomes in males (L1 and L2 autosome pairs plus X chromosome) and the six eu-chromosomes in females of the species (L1, L2 and X chromosome pairs) form polytene elements. The group of heterochromatic S autosomes and the Y chromosome appear to be absent in polytene nuclei. The information contained in this paper may further the development of genetical control methods of tsetse flies, and should assist in phylogenetic analyses of the genus Glossina.