<jats:title>Abstract</jats:title><jats:p>SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of <jats:italic>SOX9</jats:italic> expression in XY gonads. Loss‐of‐function <jats:italic>SOX9</jats:italic> variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain‐of‐function (GoF) <jats:italic>SOX9</jats:italic> variants have not been reported previously. We report the case of a 16‐year‐old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next‐generation sequencing‐based genetic screening for disorders of sex development led to the identification of a novel <jats:italic>SOX9</jats:italic> variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K‐SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype‐SOX9. This GoF activity was not observed in the luciferase reporter containing <jats:italic>Amh</jats:italic>, the gene for anti‐Müllerian hormone. We genetically engineered female mice (<jats:italic>Sox9</jats:italic><jats:sup>E50K/E50K</jats:sup>), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel <jats:italic>SOX9</jats:italic> variant with a promoter‐specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF <jats:italic>SOX9</jats:italic> variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.</jats:p>