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The shortfin mako, Isurus oxyrinchus, is an oceanic pelagic shark species found worldwide in tropical and subtropical waters. It is frequently caught by pelagic longline fisheries, but despite its commercial importance and ecological significance, little is still known about its biology and ecology at the molecular level. Therefore, we combined two massive parallel sequencing approaches, double digest restriction site-associated DNA sequencing (ddRAD) and RNA sequencing (RNAseq), for single nucleotide polymorphism (SNP) discovery in the shortfin mako. The ddRAD yielded a total of 82,676 putative SNPs. For RNAseq, a total of 129,663 putative SNPs were found. After the stricter filtering procedure, 405 SNPs from ddRAD and 1165 SNPs from RNAseq were retained and suitable for further analysis. Annotation analysis of SNPs from ddRAD revealed a total of 55 gene associated SNP markers, of which 32 SNPs (58.2%) are associated with diseases and defense responses, 9 SNPs (16.4%) are associated with developmental process, and 3 SNPs (5.4%) are present in genes involved in the reproductive function. For RNAseq, 739 SNPs were annotated and associated to relevant functions amongst which 10 SNPs (0.53%) were related with reproduction, 6 SNPs (0.32%) with growth, and 9 (0.48%) with locomotion. Overall, the genotyping of the SNPs was followed by the validation of 255 SNPs from ddRAD and 646 for RNAseq in 31 individuals from the Atlantic and Indian oceans. Our results provide valuable sequence resources for future population genomics analysis, comparative genomics, phylogenomics, and molecular evolution of the globally endangered shortfin mako shark. [ABSTRACT FROM AUTHOR]