Solea senegalensis is a fish species undergoing domestication, although many problems, such as mastering reproduction, must be solved before determining up standardized culture conditions. Both domestication and broodstock management would be served by development and screening of genetic markers. In this study, we developed a microsatellite PCR multiplex for S. senegalensis adapted to automated detection systems, made up of five loci (Sol19A, SolCA13, SolMII, Sol9A and Sol13D). Its reliability has been proved by obtaining reproducible genotyping data from a wide range of individuals.We applied these markers to evaluate the genetic structure of a fish stock of a Spanish company which presented serious spawning problems. This was performed by comparing the farm broodstock (br), its offspring (pr) and one wild sample (rf). Data revealed similar genetic structure between br and rf, and a substantial reduction, in one generation, of the genetic variability for pr relative to br and rf. A second application was parentage monitoring in the br group, for assessing the genetic processes underlying its reproduction. Moreover, the capacity of this multiplex PCR tool for pedigree tracing was evaluated under different situations by means of simulation assays.These results across all five loci indicate the suitability of the five microsatellites to be used in studies of pedigree tracing, with an efficiency higher than 98% and 90% in 10 × 10 and 20 × 20 crosses, respectively and, thus could be useful in broodstock management.Pedigree data indicated that only one female and two males had contributed to the offspring, thus generating two half-sib families. Results showed how broodstock development using F1 descendants, in absence of genetic screening, might lead to a drastic reduction in the level of genetic variability and to a strong relatedness among breeders. It is known that these practices are generally applied on many farms, thus having putative negative consequences in the current poor performance shown by S. senegalensis.