WNT family proteins transduce signals through Frizzled (FZD) receptors with extracellular WNT-binding domain and cytoplasmic Dishevelled-binding domain. WNT2B-ST7L and WNT2-ST7 loci are paralogous regions within the human genome. WNT2B isoform 2 and WNT2 are canonical WNTs to activate the beta-catenin - TCF pathway in human cancer, especially in gastric cancer. Here, the rat Wnt2b gene was identified and characterized by using bioinformatics. The rat Wnt2b gene, consisting of five exons, was located within AC106372.5 genome sequence. Wnt2b core promoter region (nucleotide position 234449-233652 of AC106372.5 rat genome sequence) was identified as an evolutionarily conserved region. GC content of rat Wnt2b promoter was 71.8%. Nucleotide identity between human and rat Wnt2b promoters was 71.2%. E box, CP2-binding site, CCAAT box and ZNFN1A1 (IKAROS)-binding site were located within the conserved region of rat Wnt2b, mouse Wnt2b, and human WNT2B promoters. Rat Wnt2b (391 aa), human WNT2B isoform 2 and other vertebrate Wnt2b orthologs were type II transmembrane proteins with cleavable extracellular WNT domain, while vertebrate Wnt2 orthologs were secreted proteins. Twenty-four Cys residues were conserved among vertebrate Wnt2b and Wnt2 orthologs. Glycosylation sites at Asn 283 of rat Wnt2b and RGD motif were identified within mammalian Wnt2b orthologs, but not within vertebrate Wnt2 orthologs and zebrafish wnt2b. Zebrafish wnt2b acquired N-terminal cytoplasmic region to evolve into type II transmembrane protein, and then mammalian Wnt2b orthologs acquired integrin-targeting RGD motif within the extracellular WNT domain. This is the first report on the rat Wnt2b gene as well as on molecular evolution of the Wnt2b orthologs.