Despite sequence information from many vertebrates the evolution of the neuropeptide Y (NPY) family of peptides has been difficult to resolve, particularly among ray-finned fishes. We have used chromosomal location and sequence analyses to identify orthologs and gene duplicates in teleost fish genomes. Our analyses support origin of NPY and peptide YY (PYY) from a common ancestor in early vertebrate evolution through a chromosome duplication. We report here that the teleost tetraploidization generated duplicates of both NPY and PYY and that all four genes are still present in the two sequenced pufferfish genomes Tetraodon nigroviridis and Takifugu rubripes as well as three-spined stickleback, Gasterosteus aculeatus. The zebrafish Danio rerio NPYb gene has probably been lost whereas medaka, Oryzias latipes seems to lack PYYb. Some of the previously published PYY sequences were misidentified and actually constitute NPYb. Our analyses confirm that the peptide previously named PY in some fish species is a duplicate of the PYY gene and hence should be called PYYb. The NPYa and NPYb genes in Takifugu rubripes are predominantly expressed in brain, as detected by RT-PCR, whereas PYYa and PYYb are expressed in several organs including brain, intestine and gonads. Thus, also the resemblance in expression pattern supports the fish gene duplication scenario. Our study shows that when sequence comparisons give ambiguous results, chromosomal location can serve as a useful criterion to identify orthologs. This strategy may help to resolve relationships in several families of short peptides.