Vertebrate species exhibit some of the most varied of sex determination systems found amongst teleost fish. Understanding evolution of species specific mechanisms can help guide development of a DNA based sex assays for fisheries management applications. This presentation describes a genomic rearrangement that gave rise to new sex chromosomes in tuna. We assembled highly complete genome sequences for all eight living true tuna species, skipjack tuna, and Spanish mackerel – a close relative of the tunas. An inter-chromosomal translocated gene duplication was found that produced a single candidate gene for sex determination, SULT1-Y. This male-specific locus was shared among all tuna species, including skipjack1. The highly conserved genetic sequence of this gene enabled development of a pan-tuna molecular test for genotypic sex. The absence of this gene from Spanish mackerel and other scombrid relatives appears to coincide with the evolutionary origin of the tuna lineage that conferred a new mode of genetic sex determination and, in doing so, may have provided a catalyst for their speciation. Development of this pan-tuna genetic sex marker has since proven to be a useful addition to the suite of techniques currently applied to genomics based fishery management of several commercially important tuna species.