Sex determination is exceptionally diverse and shows high evolutionary rate in fish. European hake (Merluccius merluccius) is a species of great commercial value distributed throughout European coasts, which displays a significant sexual growth dimorphism. We present a chromosome-level genome of M. merluccius, assembled into 215 contigs using long- and short-read sequencing, further scaffolded into the species’ 21 chromosomes using Omni-C (715 Mb). RNA-Seq on several tissues from pooled individuals improved annotation (26,625 protein-coding genes and 16,766 ncRNAs). Five males and five females from an Atlantic population were re-sequenced at 30x coverage to look for association with sex across the whole genome. Genetic differentiation between males and females (FST) and intrapopulation fixation index (FIS) pointed to a region on chromosome 9 spanning ~10 Mb which included several genes related to gonad differentiation and showed strong linkage disequilibrium associated to a putative inversion. Near sox3 (~25 kb), SNPs were mostly heterozygous in males and homozygous in females, consistent with an XX/XY SD system. These SNP markers were validated in a larger sample of 56 males and 65 females from the same population using MassARRAY. Other genomic regions that were differentiated between males and females and suggestive of sexual conflict were also explored across the genome. Results support a candidate master sex determining (MSD) gene in M. merluccius and indicate some differentiated regions potentially under sexual conflict. This information will be useful for the fisheries management of M. merluccius in the context of climate change, where non-invasive sex identification tools are essential.