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Genetic markers in tilapia species associated with loci affecting sex determination (SD), sex-specific mortality or both were mapped to linkage groups (LG) 1, 2, 3, 6 and 23. The objective of this study was to use these markers to fine-map the locus with the greatest effect on SD in Oreochromis niloticus. Our parental stock, full-sibs of Nile tilapia (Swansea origin), were divided into three groups: (i) untreated, (ii) feminized by diethylstilbestrol and (iii) masculinized by 17α-methyltestosterone. We analysed the first group for association of microsatellite markers representing these five LGs. The strongest association with gender was found on LG23 for marker UNH898 (χ2; P = 8.6 × 10−5). Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male-associated allele (MAA). Sex-reversed individuals were used for mating experiments with and without the segregating MAA. Mating of individuals lacking the MAA resulted in all-female progeny. Mating of two heterozygotes for MAA gave rise to 81 males and 30 females. Analysis of association between gender and genotypes identified the MAA in 98.6% of males as opposed to 8.0% of females (χ2; P = 2.5 × 10−18). Eight markers that flank UNH898 were genotyped to map the locus on LG23 within a confidence interval of 16–21 cM. Mating of homozygous individuals for MAA is underway for production of all-male populations.