In this study we have developed protocols for induced triploidy and gynogenesis of Senegalese sole (Solea senegalensis), a promising flatfish species for marine aquaculture, in order to: 1) identify the sex-determination mechanism; and 2) to improve its production by generating a) sterile fish, avoiding problems related with sexual maturation, and b) all-female stocks, of higher growth rate. Triploidy was induced by means of a cold shock. Gynogenesis was induced by activating eggs with UV-irradiated sperm, and to prompt diploid gynogenesis, a cold-shock step was also used. Ploidy of putative triploid larvae and gynogenetic embryos were determined by means of karyotyping and microsatellite analysis. Haploid gynogenetic embryos showed the typical "haploid syndrome". As expected, triploid and gynogenetic groups showed lower fertilization, hatching, and survival rates than in the diploid control group. Survival rate, calculated 49 days after hatching, for haploid and diploid gynogenetic groups was similar to those observed in other fish species (0% and 62.5%, respectively), whereas triploids showed worse values (45%). Sex was determined macroscopically and by histological procedures, revealing that all the diploid gynogenetic individuals were females. In conclusion, we have successfully applied chromosomal-manipulation techniques in the flatfish species Senegalese sole in order to produce triploid, haploid, and diploid gynogenetic progenies.