The overall understanding of the sex differentiation cascade in vertebrates is still growing slowly, probably because of the variety of vertebrate models used and the number of molecular players yet to be discovered. Finding conserved mechanisms among vertebrates should provide a better view of the key factors involved in this process. To this end, we used real-time reverse transcription-polymerase chain reaction to produce a temporal map of fluctuations in mRNA expression of 102 genes during sex differentiation and early gametogenesis in the rainbow trout (Oncorhynchus mykiss). We used these 102 temporal gene expression patterns as a basis for a hierarchical clustering analysis to find characteristic clusters of coexpressed genes. Analysis of some of these gene clusters suggested a conserved overall expression profile between the sex differentiation cascade in fish and mammals. Among these conserved molecular mechanisms, sox9, dmrt1, amh, nr5a1, nr0b1, igf1, and igf1ra are, for instance, characterized as early expressed genes involved in trout testicular differentiation as it is known or suggested in mammals. On the contrary, foxl2, fst, and lhr are characterized as early expressed genes during trout ovarian differentiation, as also found in mammals. Apart from this high conservation, our analysis suggests some potential new players, such as the fshb subunit gene, which is detected here for the first time, to our knowledge, in the female differentiating gonad of a vertebrate species and displays a specific overexpression that coincides in timing with the occurrence of first oocyte meioses, or the pax2 gene, which displays an early and testis-specific expression profile.