For aquaculture species that have desirable sexual dimorphic traits, manipulation of the sexual development is beneficial. For example, a male tilapia grows almost twice as fast as a female, which translates into shorter culturing time and more uniform harvest size. The commonly used method for getting an all-male tilapia population is by oral administration of the synthetic male sex hormone (17α-methyltestosterone). Alternative hormone-free masculinization method is being explored to increase the product value due to consumers preconception of 17α-methyltestosterone impacts on environment and human health. Heat-induced masculinization, which is a common phenomenon in many teleost species, is a hormone-free alternative but its underlying mechanism is unclear. Using the model organism zebrafish, we established the conditions for heat-induced masculinization and performed in-depth gonadal transcriptomic analysis. It was observed that the degree of masculinization varied among different zebrafish families, indicating interactions between the genotype and the environment (GxE). Major gonadal transcriptomic reprogramming was observed in juvenile zebrafish after exposure to elevated temperature and in some fish this effect persisted for a prolonged period of time even after temperature returned to normal. These findings in zebrafish can serve as reference for the implementation of heat-induced masculinization for aquaculture species.