Production of triploid individuals is a relevant goal for the aquaculture industry due to the benefits associated with their sterility and growth. Thus, methods for assessing triploidy have been developed based on genome, chromosome or gene triploid-associated properties. In this study, we developed a new cheap, technically simple and accurate method to validate triploidy in turbot (Scophthalmus maximus) based on microsatellite markers. Five crosses were performed to produce diploid and triploid progenies that were used to validate this molecular tool. Flow cytometry, one of the most widely used and accurate techniques for ploidy determination, was used as reference to contrast results. A set of four highly polymorphic and largely distant to centromere microsatellites was selected for this purpose. Ploidy was easily evaluated according to the maximum number of alleles at the microsatellite loci tested, diploids showing two and triploids three. These microsatellites were combined in a single multiplex and were able to identify triploids with 100% accuracy in all analyzed crosses. 2