We compared primary and secondary structures of V4 (helices E23-2 to E23-5) and V7 (helix 43) regions of 18S rRNAs in insects and the other three major arthropod groups (crustaceans, myriapods, and chelicerates) known so far. We found that the lengths of primary sequences and the shapes of secondary structures of these two hypervariable regions of insect 18S rRNA even at infraclass levels are phylogenetically informative and reflect major steps in insect evolution. The long sequence insertion and bifurcated shape of helices E23-2 to E23-5 in the V4 region are unique synapomorphic characters for winged insects (Pterygota). The long sequence insertion and expanded stem length of helix 43 in the V7 region are synapomorphic characters for holometabolous insects which conduct complete metamorphosis. The strongly conserved secondary structures suggest the possibility that these hypervariable regions may be related with certain important cellular functions unknown thus far. The comparison with insect fossil records revealed that the pterygote synapomorphy (V4) and the holometabolous synapomorphy (V7) were established prior to the acquisition of insect wings (flight system) and prior to the development of complete metamorphosis, respectively. These synapomorphies have been also relatively stable over at least 300 Myr and 280 Myr, respectively as well. It implies that the expansion events of the V4 and V7 regions have not occurred simultaneously but independently at different periods during the insect evolution. Then this suggests that V4 and V7 regions are not functionally correlated as recently suggested by Crease and Coulbourn.