[Objectives] Currently, the primary methods for determining the sex of Chinese Soft-shelled Turtle Pelodiscus sinensis include visual inspection of external features, examination of reproductive organs after dissection, and DNA PCR analysis. However, each of these methods has its limitations and cannot fully meet the requirements for sex determination in the huge production of Chinese Soft-shelled Turtle. Therefore, there is an urgent need to develop a rapid, minimally invasive and accurate method for determining the genetic sex of Chinese Soft-shelled Turtles. [Methods] In this study, blood was collected from veins of the calipash in adult Chinese Soft-shelled Turtles. The blood supernatant was used as a DNA template for PCR reaction and identification following rapid pretreatment. The feasibility of the genetic method was assessed by optimizing the pretreatment method, blood volume and PCR system (including primer concentration, template concentration, DNA polymerase mix volume and annealing temperature, among other factors), as well as by comparative blood DNA PCR tests, external morphological observations, anddissection of reproductive organs. [Results] The results demonstrated that after rapid pretreatment of 5 μl fresh turtle blood using an alkaline boiling method, a 20 μl PCR system was prepared by adding 2 μl of supernatant DNA template, 10 μl of DNA polymerase mixture, 0.8 μl each of upstream and downstream primers, and 6.4 μl of ddH2O. PCR amplification was then performed at an annealing temperature of 58 ℃, followed by detection using 1% agarose gel electrophoresis. The results revealed that female turtles (ZW type) exhibited distinct double bands with molecular sizes approximately 860 bp and 1 500 bp, while male turtles (ZZ type) exhibited a clear single band with molecular sizes around 860 bp (Fig. 1). The accuracy of sex identification in Chinese Soft-shelled Turtles reached 100%, which was confirmed by the blood DNA PCR validation test, external morphological observations and examinations of reproductive organ (Figs. 8﹣10). [Conclusion] The direct blood PCR method based on alkaline boiling pretreatment is considered a rapid and cost-effective approach for sex determination in Chinese Soft-shelled Turtles.