The flatfish Solea senegalensis represents an important resource in modern mariculture and is largely raised in South Spain and Portugal. Substantial progress has been achieved in its domestication, though suitable reproduction and zootechnical conditions still remain unknown. Difficulties to obtain breeders from wild lead many companies to set up broodstocks with first generation (G1) progeny without genetic guidelines. To study the genetic processes underlying the early stage of domestication of this species, the genetic structure of four representative broodstocks from Southern Spain has been assessed by means of eight microsatellite loci. Data revealed a substantial reduction in levels of genetic variability on just one generation in stocks totally or partially composed of G1 individuals when compared with those solely integrated by individuals from wild. The genetic relatedness within the four stocks has been established, having detected close relationships between individuals from cultured origin, thus suggesting that the loss of variability is apparently due to setting up broodstocks by blind selecting of family related G1 individuals. The high proportion of siblings in these stocks can have negative consequences in future generations due to inbreeding effects. The relationships between the four broodstocks were traced, having found a common origin between two of them, which come from the same donor hatchery, thus enhancing the homogenization of the existent resources for S. senegalensis among the various stocks. This finding alerts about the risks in exchanging fish between hatcheries without knowing their genetic origin. From this study, the use of microsatellites is strongly recommended to control the genetic composition of S. senegalensis broodstocks, aimed to maintain standards of genetic health and improve their reproduction capacity, which is a key issue in the domestication process.