Sex determination in the blue tilapia (Oreochromis aureus) is thought to be a WZ-ZZ (female heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major gene using the technique of bulked segregant analysis. We identified 11 microsatellite markers on linkage group 3 which were linked to phenotypic sex. The putative W chromosome haplotype correctly predicts the sex of 97% of male and 85% of female individuals. Our results suggest the W locus lies within a few centimorgans of markers GM354, UNH168, GM271 and UNH131. Markers on LG1 also showed a strong association with sex, and indicate the segregation of a male-determining allele in this region. Analysis of epistatic interactions among the loci suggests the action of a dominant male repressor (the W haplotype on LG 3) and a dominant male determiner (the Y haplotype on LG1). These markers have immediate utility for studying the strength of different sex chromosome alleles, and for identifying broodstock carrying copies of the W haplotype.