The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphic bands amplifying preferentially in males of the Mount Lassen and Scottish strains of rainbow trout. Chromosomal localization using fluorescent in situ hybridization of a 900 bp probe developed from one of these markers revealed a brightly defined signal on a chromosome that could morphologically be classified as the Y chromosome.