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Effective farming of tilapia requires all-male culture, characterized by uniformity and high growth rate. Males of O. aureus(Oa) and females of O. niloticus(On) produce all-male offspring, but there is a behavioral reproductive barrier between the two species that prevents mass production. In crosses between Oaand Onbroodstocks, few hybrid females are attracted to the Oamale nests (denoted responders), and if they harbor the Onalleles for the sex determination (SD) sites on linkage groups (LGs) 1, 3, and 23, all-male progeny are produced. Yet, without controlling for the alleles underlying SD, the parental stocks gradually lose their capability for all-male production. Hypothesizing that marker-assisted selection for female responders would allow production of sustainable broodstocks, we applied genotyping-by-sequencing to generate 4983 informative SNPs from 13 responding and 28 non-responding females from two full-sib families. Accounting for multiple comparisons in a genome-wide association study, seven SNPs met a false discovery rate of 0.061. Lowest nominal probabilities were on LGs 9 and 14, for which microsatellite DNA markers were designed within the candidate genes PTGDSLand CASRL, respectively. By increasing the sample size to 22 responders and 47 non-responders and by genotyping additional established microsatellites, we confirmed the association of these LGs with female responsiveness. The combined effects of microsatellites GM171and CARSL-LOC100690618on LGs 9 and 14 explained 37% of the phenotypic variance of reproductive interaction (p< 0.0001). Based on these findings, we propose a strategy for mass production of all-male tilapia hybrids through selection for genomic loci affecting SD and female responsiveness.