The Cyclostomata consists of the two orders Myxiniformes (hagfishes) and Petromyzoniformes (lampreys), and its monophyly has been unequivocally supported by recent molecular phylogenetic studies. Under this updated vertebrate phylogeny, we performed in silico evolutionary analyses using currently available cDNA sequences of cyclostomes. We first calculated the GC-content at four-fold degenerate sites (GC₄), which revealed that an extremely high GC-content is shared by all the lamprey species we surveyed, whereas no striking pattern in GC-content was observed in any of the hagfish species surveyed. We then estimated the timing of diversification in cyclostome evolution using nucleotide and amino acid sequences. We obtained divergence times of 470–390 million years ago (Mya) in the Ordovician–Silurian–Devonian Periods for the interordinal split between Myxiniformes and Petromyzoniformes; 90–60 Mya in the Cretaceous–Tertiary Periods for the split between the two hagfish subfamilies, Myxininae and Eptatretinae; 280–220 Mya in the Permian–Triassic Periods for the split between the two lamprey subfamilies, Geotriinae and Petromyzoninae; and 30–10 Mya in the Tertiary Period for the split between the two lamprey genera, Petromyzon and Lethenteron. This evolutionary configuration indicates that Myxiniformes and Petromyzoniformes diverged shortly after the common ancestor of cyclostomes split from the future gnathostome lineage. Our results also suggest that intra-subfamilial diversification in hagfish and lamprey lineages (especially those distributed in the northern hemisphere) occurred in the Cretaceous or Tertiary Periods.