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Temperature tolerance is an important trait from both an economic and evolutionary perspective in fish. Because of difficulties with measurements, genome-wide selection using quantitative trait loci (QTLs) affecting Upper temperature tolerance may be an alternative for genetic improvement. Turbot Scophthalmus maximus (L.) is a cold-water marine fish with high economic value in Europe and Asia. The genetic bases of upper temperature tolerance (UTTs) traits have been rarely studied. In this study, we constructed a genetic linkage map of turbot using simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers. A total of 190 SSR and 8,123 SNP were assigned to 22 linkage groups (LGs) of a consensus map, which spanned 3,648.29 cM of the turbot genome, with an average interval of 0.44 cM. Moreover, we re-anchored genome sequences, allowing 93.8% physical sequences to be clustered into 22 turbot pseudo-chromosomes. A high synteny was observed between two assemblies from the literature. QTL mapping and validation analysis identified thirteen QLTs which are major effect QTLs, of these, 206 linked SNP loci, and two linked SSR loci were considered to have significant QTL effects. Association analysis for UTTs with 129 QTL markers was performed for different families, results showed that eight SNP loci were significantly correlated with UTT, which markers could be helpful in selecting thermal tolerant breeds of turbot. 1,363 gene sequences were genomically annotated, and 26 QTL markers were annotated. We believe these genes could be valuable candidates affecting high temperatures, providing valuable genomic resources for the study of genetic mechanisms regulating thermal stress. Similarly, they may be used in marker-assisted selection (MAS) programs to improve turbot performance. [ABSTRACT FROM AUTHOR]