A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.