Recent studies demonstrated that sequence divergence in both transcriptional regulatory region and coding region contributes to the subfunctionalization of duplicate gene. However, whether sequence divergence in the 3′-untranslated region (3′-UTR) has an impact on the subfunctionalization of duplicate genes remains unclear. Here, we identified two diverging duplicate vsx1 (visual system homeobox-1) loci in goldfish, named vsx1A1 and vsx1A2. Phylogenetic analysis suggests that vsx1A1 and vsx1A2 may arise from a duplication of vsx1 after the separation of goldfish and zebrafish. Sequence comparison revealed that divergence in both transcriptional and translational regulatory regions is higher than divergence in the introns. vsx1A2 expresses during blastula and gastrula stages and in adult retina but silences from segmentation stage to hatching stage, vsx1A1 starts expression from segmentation onward. Comparing to that zebrafish vsx1 expresses in all the developmental stages and in the adult retina, it appears that goldfish vsx1A1 and vsx1A2 are under going to share the functions of ancestral vsx1. The different but overlapping temporal expression patterns of vsx1A1 and vsx1A2 suggest that sequence divergence in the promoter region of duplicate vsx1 is not sufficient for partitioning the functions of ancestral vsx1. By comparing vsx1A1 and vsx1A2 3′-UTR-linked green fluorescent protein gene expression patterns, we demonstrated that the 3′-UTR of vsx1A1 remains but the 3′-UTR of vsx1A2 has lost the capability of mediating bipolar cell specific expression during retina development. These results indicate that sequence divergence in the 3′-UTRs has a clear effect on subfunctionalization of the duplicate genes. J. Exp. Zool. (Mol. Dev. Evol.) 318B:531–544, 2012. © 2012 Wiley Periodicals, Inc.