Gilthead seabream (Sparus aurata L.) is one of the most important species in the Mediterranean aquaculture. The industry has optimized their productions through mainly management factors such as nutrition, reproduction or prevention of disease, but not by genetic improvement. This is due in part to that the reconstruct the genealogy of fish represents a high cost for companies. This is because culture of this species is generally performed through mass spawning where parent–offspring relationships are unknown, so the introduction of breeding programs in this kind of industrial production system requires physical tagging to identify all individuals in conjunction with DNA analysis to parental assignments. All this considered, the general aim of this work is the development of two new multiplex PCRs for gilthead seabream with specific microsatellites as useful genotyping tool for parental assignments of offspring from controlled mass-spawning without interfering with the production system. At the same time, these multiplex PCRs could also be used in QTL determination, and MAS selection.