In humans, the claudin superfamily consists of 19 homologous proteins that commonly localize to tight junctions of epithelial and endothelial cells. Besides being structural tight-junction components, claudins participate in cell–cell adhesion and the paracellular transport of solutes. Here, we identify and annotate the claudin genes in the whole-genome of the teleost fish, Fugu rubripes (Fugu), and determine their phylogenetic relationships to those in mammals. Our analysis reveals extensive gene duplications in the teleost lineage, leading to 56 claudin genes in Fugu. A total of 35 Fugu claudin genes can be assigned orthology to 17 mammalian claudin genes, with the remaining 21 genes being specific to the fish lineage. Thus, a significant number of the additional Fugu genes are not the result of the proposed whole-genome duplication in the fish lineage. Expression profiling shows that most of the 56 Fugu claudin genes are expressed in a more-or-less tissue-specific fashion, or at particular developmental stages. We postulate that the expansion of the claudin gene family in teleosts allowed the acquisition of novel functions during evolution, and that fish-specific novel members of gene families such as claudins contribute to a large extent to the distinct physiology of fishes and mammals.