A cDNA encoding for a novel rainbow trout SHBG was identified and characterized. Phylogenetic analysis showed that this novel SHBG, named SHBGb, was a highly divergent paralog of the classical SHBG (SHBGa) form previously known in vertebrates including zebrafish, seabass, and rainbow trout. Using all available sequences, no SHBGb-like sequence could be identified in any fish species besides Atlantic salmon. Rainbow trout SHBGa and SHBGb share only 26% sequence identity at the amino acid level and exhibit totally distinct tissue distribution, thus demonstrating a functional shift of SHBGb. Indeed, shbga mRNA was predominantly expressed in liver and spleen but could not be detected in the ovary, whereas shbgb had a predominant ovarian expression but could not be detected in liver. Despite its high divergence, rainbow trout SHBGb expressed in COS-7 cells could bind estradiol and testosterone with high affinity and specificity. Both rainbow trout shbgb mRNA and proteins were localized to the granulosa cells of vitellogenic ovarian follicles, whereas SHBGb immunoreactivity was also found in theca cells. Finally, shbgb ovarian mRNA expression exhibited a significant drop between late vitellogenesis and oocyte maturation at a time when ovarian aromatase (cyp19a) gene expression and estradiol circulating levels exhibited a dramatic decrease. Together, these observations show that SHBGb is a functional and highly divergent SHBG paralog probably arising from a salmonid-specific duplication of the shbg gene.