Sex determination (SD) mechanisms are exceptionally diverse and show high evolutionary rate in fish. At least 21 different SD genes have been consistently identified in more than 100 fish species to date. Exploring the i ntraspecific variation on the genes underlying SD in fish genomes as well as the environmental factors influencing sex could aid to understand this huge dynamism. Furthermore, sexual conflict regions that have been claimed as one of the initial triggers on new SD mechanisms should be inspected more extensively in fish genomes to identify potential candidate regions facilitating the transition to a new SD system. The European hake ( Merluccius merluccius ) is a species of great commercial value distributed throughout European coasts, which displays a significant sexual growth dimorphism and that has also been identified as a potential target for aquaculture diversification. Here, we present the first chromosome-level genome of the European hake assembled through long- and short-read sequencing. The genome was initially assembled in 162 scaffolds ( N50: 32 Mb) that were anchored using Hi-C into 21 chromosomes spanning 715 Mb. Repetitive elements represented 43% of the genome. We performed RNA-Seq on muscle, liver, brain, spleen and gonad from pools of individuals along with proteomes from closely related species and gene predictors for improving annotation ( 26,625 protein-coding genes and 41,543 isoforms ; 11,083 sncRNAs and 5,683 lncRNAs) . Five males and five females were re-sequenced using 30X coverage 150 bp PE Illumina sequencing for sex-association genome screening to identify the SD region . Around 12 million SNPs were consistently called and, after filtering by MAF > 0.3, read depth (10 > x > 300) and no missing data in the 10 individuals, a total of 1,552,561 SNPs were retained for genome screening (1 SNP / 5.6 kb) . We estimated genetic differentiation (FST) and intrapopulation fixation index (FIS ) using male and female populations across windows of 20 SNPs to identify candidate SD regions (for a typical XY/ZW SD system: FST = 0.33 and FIS = -1.0). Chromosome 9 showed a significant differentiation between males and females between 10 and 20 Mb, which included several genes related to gonadal and sex differentiation (ar , pgr , fox4 , foxN3 , tex11 , sox3 , mospd2 and nr3c1). After zooming, we identified a candidate region spanning 7 kb very close to sox3 (20 kb distance), where most SNPs were heterozygous in males and homozygous in females compatible with a XX/XY system. Sex association of these markers was validated in a large sample of 45 males and 45 females using a MassARRAY genotyping platform. Further, several regions under putative sexual conflict were identified in the hake genome, and one of them in chromosome 5, including clusters of immune- and reproduction-related genes, was validated in the larger sample . This information supports a candidate gene for SD in European hake and provide new insights on fish SD through inspection of sexual conflict regions. Finally, a SD molecular tool could be derived from our data useful for fisheries management of European hake in a context of climatic change.