Abstract: Visual system homeobox-1 ( vsx1) is a homeobox gene encoding a homeodomain and a CVC domain that was first cloned from an adult goldfish retinal library. This gene is expressed in different regions and tissues of the embryo during development. It plays a pivotal role in differentiation and maintenance of retinal bipolar cells. To study the regulatory mechanism of vsx1 expression, we analyzed the genomic structure and intron polymorphism of goldfish vsx1 by PCR. The gene was found to consist of five exons and four introns. The exon-intron structure is conserved among zebrafish vsx1, mouse vsx1 and human vsx1. Two obviously different categories were determined in the first intron of goldfish vsx1, one has 39 base pairs, which contain a core sequence of eukaryote enhancer, more than the other. No significant divergence was detected in the other three introns. The results suggest that the first intron of goldfish vsx1 may be associated with the expression regulatory of vsx1. The significant polymorphism in the first intron of goldfish vsx1 also provides us with suitable probes to analyze expression levels of vsx1 at the same loci or different locus as well as the regulatory mechanism of vsx1 tissue-specific expression.