Through phylogeny reconstruction we identified 49 genes with a single copy in man, mouse, and chicken, 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 had orthologs in the pufferfish (Takifugu rubripes). For another 20 of these genes, we found only one pufferfish ortholog but in each case it was more closely related to one of the zebrafish duplicates than to the other. Forty-three pairs of duplicated genes map to 24 of the 25 zebrafish linkage groups but they are not randomly distributed; we identified 10 duplicated regions of the zebrafish genome that each contain between two and five sets of paralogous genes. These phylogeny and synteny data suggest that the common ancestor of zebrafish and pufferfish, a fish that gave rise to ∼22,000 species, experienced a large-scale gene or complete genome duplication event and that the pufferfish has lost many duplicates that the zebrafish has retained. [Supplemental material is available online at www.genome.org.