This study aimed to determine the optimum dosage and duration of diethylstilbestrol hormone (DES) required to produce the highest percentage of females and the use of short sequence repeats (SSRs) markers for the sexing of the Nile tilapia (Oreochromis niloticus). Fries with a total length of 9-11mm were used. DES hormone was added to the fries' feed in two concentrations (100 and 200mg/ kg of feed) for 28 and 40 days, excluding the control feed (without DES). To differentiate between normal females (XX) and sex-reversed females (XY), progeny test and SSRs markers were used. The hormonal treatment of 200mg/kg feed for 28 and 40 days gave the highest females percentage, with a slight difference between them (85.51 and 86.54, respectively). While, the treatment for 28 days gave a higher percentage of survival than 40 days (23,00 and 17.33, respectively). From 22 tested females, three XY females were obtained by progeny test and SSRs markers. Production of sex-reversed females can be used as a first step for the production of supper male YY of the Nil tilapia, and SSRs markers can be considered as a promising molecular marker for sex determination that can be used to reduce the duration of the Nile tilapia breeding program.