Steroid hormones; androgen and estrogen are thought to be the primary drivers of the tilapia sex differentiation (male and female). Cytochrome P450 aromatase is a steroidogenic enzyme, which changes androgens into estrogens and mediates the biosynthesis of estrogens. The current study investigated the aromatase immunoreactivity in the Nile tilapia's (Oreochromis niloticus) gonads larvae under temperature-induced and sex differentiation that occurs naturally. When the tilapia larvae were subjected to 35°C of high temperatures, the majority of them produced significantly more males (84%) than the controls (38%) at 25°C. The immunoreactivity of the enzyme aromatase was higher during normal ovarian differentiation than during testicular differentiation. This was observed in gonads subsequent to the commencement of ovarian differentiation, which involves the increase in both stromal and germ cells. In all temperature-exposed cases, there was a decline in aromatase immunoreactivity. Temperature caused females to become more masculine to varying degrees in their offspring. Additionally, the aromatase immunoreactivity decreased in males at 35°C. This shows that, for the purpose of drive differentiation toward testis development, aromatase repression within the gonad is necessary. Aromatase immunoreactivity was detected in the cytoplasm of germ cells and, to a lesser degree, in adjacent Leydig cells, following the start of differentiation in testis. This observation suggests that the aromatase immunoreactivity could be involved in the paracrine regulation of spermatogenesis. In conclusion, the temperature endocrine control for the tilapia sex differentiation shown in this research should help produce all-male populations of this fish in a way that is environmentally friendly, safe, easy to understand, and acceptable.