Developing sound breeding programs for aquaculture species may be challenging when matings cannot be controlled due to communal spawning. We developed a genotyping-by-sequencing marker panel of 300 SNPs for parentage testing and sex determination by using data from an in-house reference genome as well as a 90 K SNP genotyping array based on different populations of yellowtail kingfish (Seriola lalandi). The minimum and maximum distance between adjacent marker pairs were 0.7 Mb and 13 Mb, respectively, with an average marker spacing of 2 Mb. Weak evidence of the linkage disequilibrium between adjacent marker pairs was found. The results showed high panel performance for parental assignment, with probability exclusion values equaling 1. The rate of false positives when using cross-population data was null. A skewed distribution of genetic contributions by dominant females was observed, thus increasing the risk of higher rates of inbreeding in subsequent captive generations when no parentage data are used. All these results are discussed in the context of breeding program design, using this marker panel to increase the sustainability of this aquaculture resource.