Sex determination is a flexible process in fish, controlled by genetics or environmental factors or a combination of both depending on the species. Revealing the underlying molecular mechanisms may have important implications for research on reproductive development in vertebrates, as well as sex-ratio control and selective breeding in fish. Phenotypic sex in rainbow trout is primarily controlled by a XX/XY male heterogametic sex determination system. Unexpectedly in genetically XX all-female farmed populations, a small proportion of males or intersex individuals are regularly observed. This spontaneous masculinisation is a highly heritable trait, controlled by minor sexmodifier genes that remain unknown, although several QTL regions were detected in previous studies. In this work we used genome-based approaches and various statistical methods to investigate these QTL regions. We validated in six different French farmed populations DNA markers we had previously identified in a different commercial population on chromosomes Omy1, Omy12 and Omy20. We also identified functional candidate genes located that may be involved in spontaneous masculinisation by reducing germ cell proliferation and repressing oogenesis of XXrainbow trout in the absence of the master sex determining gene. In particular, syndig1, tlx1 and hells on Omy1, as well as khdrbs2 and csmd1 on Omy20 deserve further investigation as potential sex-modifier genes to precise their functional roles as well as their interaction with rearing temperature. Those findings could be used to produce allfemale populations that are preferred by farmers due to a delayed maturation of females and higher susceptibility of male trout to diseases.