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Ribosomal genes were mapped in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. m. submorsitans, G. p. palpalis, G. pallidipes and G. tachinoides on mitotic chromosomes by fluorescence in situ hybridization (FISH) using Drosophila hydei genomic clones that contain the 28S ribosomal DNA. In all species except G. brevipalpis, the ribosomal genes were located on the long arm of autosome L1. The Y chromosomes of G. pallidipes and G. p. palpalis showed additional hybridization signals. Supernumerary chromosomes were found in G. austeni, G. brevipalpis and G. pallidipes. The C-banding pattern obtained by in situ hybridization was compared with Giemsa C-banding patterns that were published previously. The karyotype of G. brevipalpis was found to differ from that of other Glossina species, with either two or three FISH signals being obtained with a ribosomal probe, depending on the individual analysed. The rDNA genes are the first physically mapped markers in tsetse flies and will be useful for mapping approaches.