Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD) affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid, lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as teleost fishes, amphibians, reptiles, and marsupials.