Animals choose reproductive partners based on their sexual preferences which are established at a certain time point before, during, or after sexual maturation. The preferences are often divergent within a species, which suppresses gene flow between populations and may promote speciation. There are two strains of medaka (Oryzias latipes) that differ by a single transgene and mate assortatively depending on skin color. Here, we demonstrate that symmetrically biased (mutually exclusive) sexual preferences are (1) gradually established during growth depending on skin color and the color of surrounding fish, (2) strong enough to minimize gene flow between the strains at a population level, and (3) inflexibly retained after sexual maturation, even after weeks of daily mating with partners of the other strain. Thus, these laboratory strains of medaka are under premating isolation with the simplest genomic structure. They provide an empirical platform for assessing the complex and hypothetical mechanisms of speciation by mate choice.