Up to now, temperature sex differentiation has been demonstrated in a single fish species, Menidia menidia. This study analyzes this hypothesis in Oreochromis niloticus. Treatments at 36 °C, covering the hormone sensitive period, increased the proportion of males (33% to 81%).The male proportion was affected by treatments starting not later than 13 days after fertilization and lasting 10 days or more. Survival rates were not affected by treatments. Functional masculinization of genetic females was demonstrated by the sex-ratio analysis of 10 temperature-treated males. Sex reversal by temperature treatment was also confirmed on a genotypic female population. The response to temperature strongly depends upon the breeding pairs. Hight temperatures override sex chromosome influence in Oreochromis niloticus. Therefore, Oreochromis niloticus sex is determined by genetic factors, temperature level, and genotype-temperature interactions. Temperature-dependent sex differentiation may occur more frequently in fish than previously thought, despite the presence of functional sex chromosomes.