Abstract: One of the serious problems in aqua-cultural yellow catfish is that the background of female parents is confused. It is particularly important to establish all-female families for the all-male and hybrid yellow catfish production, in which sex-reversal of XX female to XX male is a key step. Development of sex chromosome-linked markers provides a technical support for the identification of XX males in yellow catfish. In this study, different concentrations of 17α-methyltestosterone (MT) and aromatase inhibitor letrozole (LZ) were used for 54 days to treat yellow catfish (7 to 60 days post fertilization). The survival rate, body length, and body weight of each group were measured at 61 days after fertilization (61 days post fertilization). Gonadal structure was observed and histologically analyzed in XX individuals that were identified by sex-linked markers. As a result, MT and LZ treatments had no significant effects on the survival rate when compared with the control group. In addition, the XX gonad treated by MT showed seminal vesicles and could not be reversed into functional testis. However, appropriate dose of LZ could induce the sex-reversal of XX female to normal XX male, and the induction effect was enhanced with the increase of dose as well. The XX male could normally mate with XX female and YY female, which demonstrated appropriate reproductive ability. The present study proposed a reliable method to create XX male using the aromatase inhibitor LZ, which lays the foundation for the breed improvement of all-male yellow catfish and cultivation of new varieties of yellow catfish.