The study identifies 49 genes with single copies in humans, mice, and chickens, one or two copies in the tetraploid frog Xenopus laevis, and two copies in zebrafish (Danio rerio). For 22 of these genes, both zebrafish duplicates have orthologs in the pufferfish (Takifugu rubripes). Another 20 genes have one pufferfish ortholog each, but these are more closely related to one zebrafish duplicate than the other. Phylogenetic and synteny data suggest that a large-scale gene or complete genome duplication event occurred in the common ancestor of zebrafish and pufferfish, leading to the retention of many duplicates in zebrafish that were lost in pufferfish. This event likely occurred more than 500 million years ago, before the divergence of ray-finned and lobe-finned fishes but after the divergence of teleosts. The findings support the hypothesis that genome duplication and speciation are causally linked, with the potential for new gene functions and divergent resolution contributing to speciation.The study identifies 49 genes with single copies in humans, mice, and chickens, one or two copies in the tetraploid frog Xenopus laevis, and two copies in zebrafish (Danio rerio). For 22 of these genes, both zebrafish duplicates have orthologs in the pufferfish (Takifugu rubripes). Another 20 genes have one pufferfish ortholog each, but these are more closely related to one zebrafish duplicate than the other. Phylogenetic and synteny data suggest that a large-scale gene or complete genome duplication event occurred in the common ancestor of zebrafish and pufferfish, leading to the retention of many duplicates in zebrafish that were lost in pufferfish. This event likely occurred more than 500 million years ago, before the divergence of ray-finned and lobe-finned fishes but after the divergence of teleosts. The findings support the hypothesis that genome duplication and speciation are causally linked, with the potential for new gene functions and divergent resolution contributing to speciation.