The WNT16 gene at human chromosome 7q31.31 encodes two isoforms due to alternative splicing of an alternative promoter type. Here, we identified and characterized the rat Wnt16 and zebrafish wnt16 genes using bioinformatics. The rat Wnt16 and zebrafish wnt16 genes, consisting of four exons, were located within the AC117014.4 and CR925699.1 genome sequences, respectively. Exons 1b, 2, 3 and 4 of the human WNT16 gene were conserved in the rodent Wnt16 and zebrafish wnt16 genes; however, exon 1a of the human WNT16 gene was not conserved. Human WNT16 isoform 1 (NM_057168.1), consisting of exons 1b, 2, 3 and 4, was identified as the evolutionarily conserved representative isoform. Because zebrafish NM_207096.1 was an aberrant wnt16 cDNA with a retained 3'-part intron 1, complete CDS of representative zebrafish wnt16 was determined by deleting nucleotide position 183-239 from NM_207096.1 aberrant cDNA. Rat Wnt16 (364 aa) with an N-terminal signal peptide, 24 Cys residues and 3 Asn-linked glycosylation sites showed 97.3, 90.4 and 65.9% total-amino-acid identity with mouse Wnt16, human WNT16, and zebrafish wnt16, respectively. Phylogenetic analyses on WNT family members revealed that WNT16 was most closely related to WNT7A and WNT7B paralogs. Promoters of human WNT16, rat Wnt16 and mouse Wnt16 genes were well conserved. Double CCAAT motifs were conserved among mammalian Wnt16 promoters. This is the first report on the rat Wnt16 and zebrafish wnt16 genes, as well as the double CCAAT motifs within the core promoter regions of mammalian Wnt16 orthologs.