The significance of whole-genome duplications (WGD) for vertebrate evolution remains controversial, in part because the mechanisms by which WGD contributed to functional evolution or speciation are still incompletely characterized. Fish genomes provide an ideal context in which to examine the consequences of WGD, because the teleost lineage experienced an additional WGD soon after divergence from tetrapods and because five teleost genomes are available for comparative analysis. Here we present an integrated approach to characterize these post-duplication genomes based on genome-scale synteny, phylogenetic, temporal, and spatial gene expression and on protein sequence data. A minimum of 3%-4% of protein-coding loci have been retained in two copies in each of the five fish genomes, and many of these duplicates are key developmental genes that function as transcription factors or signaling molecules. Almost all duplicate gene pairs we examined have diverged in spatial and/or temporal expression during embryogenesis. A quarter of duplicate pairs have diverged in function via the acquisition of novel protein domains or via changes in the subcellular localization of their encoded proteins. We compared the spatial expression and protein domain architecture of zebrafish WGD-duplicates to those of their single mouse ortholog and found many examples supporting a model of neofunctionalization. WGDduplicates have acquired novel protein domains more often than have single-copy genes. Post-WGD changes at the gene regulatory level were more common than changes at the protein level. We conclude that the most significant consequence of WGD for vertebrate evolution has been to enable more-specialized regulatory control of development via the acquisition of novel spatiotemporal expression domains. We find limited evidence that reciprocal gene loss led to reproductive isolation and speciation in this lineage.