In spite of the numerous studies performed in the aquacultured fish gilthead seabream, the overall structure of wild and cultivated stocks is rather confusing. In this study, we developed a 10 microsatellite genotyping tool SaGT(6+4), conformed by two polymerase chain reaction-multiplex reactions (SaGT6 and SaGT4), which can be simultaneously combined in automatic sequencers. The utility of this tool was proven through the following applications: (i) characterization and differentiation of wild and cultivated populations; (ii) pedigree reconstruction and estimation of the effective size in a cultivated stock; (iii) ability for pedigree reconstruction under different simulated situations; and (iv) determination of genetic relationships in the absence of data from parents. Based on our results, some recommendations have been provided on the management of the screened stocks. Our results also support the use of this tool in a standardized way, to understand the actual status of gilthead seabream from both wild and cultivated populations.