Although the 5.8S ribosomal RNA (rRNA) of most eucaryotes consists of 155-170 nucleotides, in two dipterous species the 5.8S rRNA consists of two pieces, 123 and 30 nucleotides in length. The distribution of split 5.8S rRNA was studied in other Diptera and the most closely related order Siphonaptera to learn the origin of split 5.8S rRNA. Four species of mosquitoes, Culex tritaeniorhynchus, Culex pipiens molestus, Aedes albopictus, Anopheles sp. (Culicidae) had a single 5.8S rRNA consisting of approximately 154 nucleotides. A flea Ctenocephalides felis (Siphonaptera: Pulicidae) also had a single RNA of approximately 158 nucleotides. A crane fly (Tipulidae). a midge Orthocladius akamusi (Chironomidae), a robber fly (Asilidae), and a house fly Musca domestica (Muscidae), on the other hand, had divided 5.8S rRNA as did a fruit fly Drosophila melanogaster (Drosophilidae) and a dark-winged fungus gnat Sciara coprophila (Sciaridae). Three hypotheses are proposed on the relationship between the evolution of the 5.8S rRNA and the phylogeny of Diptera.