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Background: In Nile tilapia sex determination is governed by a male heterogametic system XX/XY either on LG1 or LG23. The latter carries a Y-specific duplicate of the amh gene, which is a testis-determining factor. Allelic variants in the amh gene demonstrated to be major triggers for autosomal and temperature-dependent sex reversal. Further, QTL on LG23 and LG20 show a temperature-responsiveness with influence on the phenotypic sex relative to the sex chromosomes. Here we present a ddRADseq based approach to identify genomic regions that show unusual large differentiation in terms of fixation index (FST) between temperature-treated pseudomales and non-masculinized females using a comparative genome-scan. Genome-wide associations were identified for the temperature-dependent sex using a genetically all-female population devoid of amh-ΔY. Results: Twenty-two thousand three hundred ninety-two SNPs were interrogated for the comparison of temperaturetreated pseudomales and females, which revealed the largest differentiation on LG23. Outlier FST-values (0.35-0.44) were determined for six SNPs in the genomic interval (9,190,077-11,065,693) harbouring the amh gene (9,602,693- 9,605,808), exceeding the genome-wide low FST of 0.013. Association analysis with a set of 9104 selected SNPs confirmed that the same genomic region on LG23 exerts a significant effect on the temperature-dependent sex. Conclusions: This study highlights the role of LG23 in sex determination, harbouring major determinants for temperature-dependent sex reversal in Nile tilapia. Furthermore FST outlier detection proves a powerful tool for detection of sex-determining regions in fish genomes. [ABSTRACT FROM AUTHOR]