[Objectives] The Chinese Giant Salamander Andrias davidianus is the largest amphibian on earth. Although its captive population has exceeded 10 million individuals, it is listed as Critically Endangered on TheIUCN Red Listof Threatened Species. Sexual identification of giant salamanders has long been challenging, although it is crucial to captive breeding and conservation. Consequently, the development of molecular markers targeting female-specific DNA fragments could offer a dependable method for distinguishing between males and females. [Methods] In this study, 15 5-month-old and 17 17-month-old individuals from an artificial breeding farm in Hanzhong, Shaanxi were randomly selected and dissected. The gonad was utilized for tissue section and Hematoxylin and Eosin staining. DNA was extracted from each individual and the female-specific DNA fragments were amplified using four pairs of primers. [Results] The primers adf225 and adf340 yielded the best amplification results, which were consistent with the morphological analysis of gonads (Fig. 1). In both male and female 5-month-old juveniles, the gonads were undifferentiated, appearing as slender, semi-transparent stripes (Fig. 2a, b). In contrast, 17-month-old juveniles displayed obvious differentiation in gonads, with granular ovaries or smooth, and white testes (Fig. 2c, d). Tissue section analysis revealed no significant morphological differences in germ cells between male and female 5-month-old individuals (Fig. 3a, b). However, in the 17-month-old individuals, ovarian cells had differentiated into larger oocytes, while still maintaining the primordial follicles (Fig. 3c). Additionally, sperm nests of testes had differentiated into spermatogonia lobules composed of spermatogonia and sertoli cells (Fig. 3d). Interestingly, there were no visible morphological differences between male and female 5-month-old or 17-month-old juveniles, indicating that external morphology alone is insufficient for sex determination (Fig. 4). [Conclusion] This study successfully identified molecular primers that are highly effective in determining the gender of juveniles. This significant finding provides a practical solution to the challenges posed by sex allocation and holds promising implications for the conservation and management of this species.