Background Sex determination processes vary widely among different vertebrate taxa, but no group offers as much diversity for the study of the evolution of sex determination as teleost fish. However, the knowledge about sex determination gene cascades is scarce in this species-rich group and further difficulties arise when considering hybrid fish taxa, in which mechanisms exhibited by parental species are often disrupted. Even though hybridisation is frequent among teleosts, gene based approaches on sex determination have seldom been conducted in hybrid fish. The hybrid polyploid complex of Squalius alburnoides was used as a model to address this question. Methodology/Principal Findings We have initiated the isolation and characterization of regulatory elements (dmrt1, wt1, dax1 and figla) potentially involved in sex determination in S. alburnoides and in the parental species S. pyrenaicus and analysed their expression patterns by in situ hybridisation. In adults, an overall conservation in the cellular localization of the gene transcripts was observed between the hybrids and parental species. Some novel features emerged, such as dmrt1 expression in adult ovaries, and the non-dimorphic expression of figla, an ovarian marker in other species, in gonads of both sexes in S. alburnoides and S. pyrenaicus. The potential contribution of each gene to the sex determination process was assessed based on the timing and location of expression. Dmrt1 and wt1 transcripts were found at early stages of male development in S. alburnoides and are most likely implicated in the process of gonad development. Conclusions/Significance For the first time in the study of this hybrid complex, it was possible to directly compare the gene expression patterns between the bisexual parental species and the various hybrid forms, for an extended set of genes. The contribution of these genes to gonad integrity maintenance and functionality is apparently unaltered in the hybrids, suggesting that no abrupt shifts in gene expression occurred as a result of hybridisation.
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