We compared gender dysphoria (GD) patients and their same-sex siblings in terms of their 2D:4D ratios, which may reflect prenatal exposure to androgen, one of the possible etiological mechanisms underlying GD. Sixty-eight GD patients (46 Female-to-Male [FtM]; 22 Male-to-Female [MtF]), 68 siblings (46 sisters of FtMs; 22 brothers of MtFs), and 118 heterosexual controls (62 female; 56 male) were included in the study. FtMs were gynephilic and MtFs were androphilic. We found that 2D:4D ratios in the both right hand (p < .001) and the left hand (p = .003) were lower in male controls than in female controls. Regarding right hands, FtM GD patients had lower 2D:4D ratios than female controls (p < .001) but their ratios did not differ from those of their sisters or male controls. FtM GD patients had no significant difference in their left-hand 2D:4D ratios compared to their sisters or female and male controls. While there was no significant difference in right hands between FtM’s sisters and male controls, left-hand 2D:4D ratios were significantly higher in FtM’s sisters (p = .017). MtF GD patients had lower right-hand 2D:4D ratios than female controls (p <.001), but their right-hand ratios did not differ from those of their brothers and male controls. There was no significant difference in left-hand 2D:4D ratios between MtF GD patients, and their brothers, or female and male controls. FtM GD patients showed significantly masculinized right-hand 2D:4D ratios, while there was no evidence of feminization in MtF GD patients.