Abstract: This study aimed to breed and identify XY female mice, and analyze their reproductive ability and the mechanism of sexual inversion, so as to provide an animal model for studying on gender determination during mouse development. The identification method of XY female mice was divided into two steps(phenotypic and genotypic identification). The first, phenotypic identification:B6.XY^TIR male was naturally mated with wild-type XX female mice, their offspring were analyzed. Male and female mice of 30-, 60- and 90-day-old were identified by external genital characteristics and anogenital distance, and the gender of fetuses of 17.5 days post coitum were identified by gonadal morphology and germ cell types. The second, genotype identification:In order to determine their sex chromosome as XX or XY, DNA of postnatal and fetal mice was extracted, primers were designed and Zfy gene (a unique gene on the Y chromosome) was amplified. Combined with phenotypic identification, B6.XY^TIR female mice were obtained. In this study, whole-tissue immunofluorescence staining was used to analyze the differentiation of gonads in fetal stage, and morphological observation was used to observe the internal reproductive system of postnatal mice, and the sequence of Zfy gene was preliminarily analyzed by sequencing. A type of XY sex reversed female mouse was obtained through phenotypic and genotypic identification. It was found by observation that the ovaries, fallopian tubes and uterine structures of the XY female mice were similar to those of wild-type XX female mice. Meanwhile, the preliminary study showed that the distribution of germ cells during gonad differentiation in XY female mice was similar to that in the wild-type XX female. Sequencing showed that only Zfy-1 gene, but not Zfy-2 gene, was amplified in B6.XY^TIR males and B6.XY^TIR females; XY females accounted for 47.47% of all 99 XY mice. The results showed that XY females had a complete female reproductive system, and sex reversed had already been formed during embryonic development. Zfy-2 might be missing on the Y chromosome of B6.XY^TIR female mice, which could be related to the occurrence of sexual inversion. The results provides a unique perspective for the further study of gonadal differentiation and gametogenesis.