Sexual reproduction is one of the most highly conserved processes in evolution and implicates many fields of biology but also animal and plant breeding and human health and society. The genetic and cell biological mechanisms making the decision whether the undifferentiated gonad of the embryo develops either towards male or female are manifold and quite different. Sex determining (SD) mechanism range from environmental to simple or complex genetic mechanisms and have evolved obviously repeatedly and independently. In species with genetic monofactorial sex determination, master SD genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program that ultimately leads to testicular or ovarian differentiation. So far very few sex determining genes have been identified in fish and in animals in general. Fish are uniquely suited to study the evolution of sex determination and SD genes. Comprising about half of the about 60 000 species of vertebrates, fish show also the greatest variety of sex determination mechanism including species with either environmental or genetic sex-determination. Even those few known SD genes are apparently not conserved over a larger number of related orders, families, genera or even species. This frequent evolutionary turnover of SD genes may be explained by the large diversity of these master sex regulators due to the high turnover of fish sex chromosomes. This project has then two major objectives: (1) to screen for potential sex determining genes in many fish species; (2) to demonstrate the role of identified genes as master sex determinants in a few selected species. To address these questions, we have developed a strategy (Rad-Sex) that makes use of next generation sequencing technology to identify genetic markers that define sex specific segments of the male or female genome. The obtained markers will be used to isolate candidates for SD genes from these regions. This approach will be carried out by screening in a first round 35 species representing major branches of the fish tree of life. Rad-Sex markers will identify sex chromosomal regions in the majority of species. From these we will then take the most promising 15 species to find candidate SD genes by screening large insert libraries with the Rad-Sex markers. Isolated clones will be sequenced and analyzed for coding sequences. Candidate genes will be evaluated by transcript profiling and finally a selection of three genes will be taken to functional characterization. The finding of new master SD genes will be of major importance taking into account the very low number of species, in which such genes have been characterized in vertebrates. From a comparative point of view, this project will also have a great impact on what is already known about the evolution of genetic sex determination in vertebrates in general and more specifically in fishes, in which the questions about the evolutionary significance of the high diversity of master sex determining genes and the genomic mechanisms allowing these rapid turnovers are largely unanswered. This project will also have important practical outcomes for molecular sexing that will be important for a better control of sex determination in aquaculture and for ecology and ecotoxicology research in some of the species investigated in this project that are economically or environmentally very important species.