The aim of this study is to develop YY supermale production of Nile Tilapia (Oreochromis niloticus) and mass production of all-male offspring on a commercial scale by crossing YY supermales with normal females. In this study, normal male genotype (XY) and supermales (YY) were treated with a dosage of 100 mg per kg food of 17α-EE for a duration of 60 days; 93.6% sex reversal females (XY△♀) and 46.88% sex reversed superfemales (YY△♀) were obtained, respectively. This result demonstrated that the normal males were far easier than supermales in sexual reversal. The progeny testing procedure results of the cross between neofemales and normal males presented 25% supermales; if YY sex reversed females mated with normal males, it resulted in half XY males and half YY males. Furthermore, when the YY sex reversed females crossed with the YY supermales, we found that all supermales presented in progeny. All of these results indicated that the Nile tilapia female has homogamety genotype XX, while the male is heterogamety XY. The progeny testing demonstrated that all crosses between YY supermales to XX females, sex reversed supermales to normal males, and sex reversed YY females to YY supermales do not result in 100% male offspring. This indicated that sex determination in Nile tilapia is not only based on the monofactorial gene but on other factors as well, including the autosomal gene and environmental factors, particularly temperature. These genetic and environmental factors combine in a rather complex way to influence the sex determination of Nile tilapia. Finally, this study found that the YY supermales give a high percentage of male offspring with 97.08%, which is crossed to normal female (XX) Nile tilapia; this result was higher than in any other previous report. Therefore, mass production of YY male broodstock and application in all male Nile tilapia culture on a commercial scale is possible.