We have determined the complete sequences of 5S rRNAs from a lamprey (Lampetra reissneri), a lancelet (Branchiostoma belcheri), silkworms (Philosamia cynthia ricini, Bombyx mori, Antheraea pernyi), and a silkworm hybrid (artificially fertilized hybrid species of Philosamia cynthia ricini male x Bombyx mori female), as well as those of cotton seeds (Gossypium hirsutum L.). Having compared more than 170 eukaryotic 5S rRNAs of which seven sequences have been determined by our group as mentioned above, we have found that the "evolutionary sites" that exist at special locations in these structures are closely related to the evolution of eukaryotes. The changes proceed step by step in an orderly way, i.e., the change in nucleotide residues of the "evolutionary sites" depends on the order of the evolution of the species and shows group-specific patterns.