Objectives: In pediatrics, anogenital distance (AGD) serves as a bioassay of normal phenotypic androgen action on the external genitalia. Recently, hypospadias and cryptorchidism were reported to be associated with reduced AGD. No normal reference ranges exist for fetuses throughout gestation. This study defines the normal centile chart of the AGD, in male and female fetuses, between 20 and 35weeks of gestation. Methods: Participants were low-risk pregnant women, 20 to 35weeks of gestation. All fetuses were singleton, with normal anatomic scan and appropriate fetal biometry for gestational age. Fetal AGD was measured by transabdominal ultrasound. The perineum was assessed in the axial plane. The distance was measured from the center of the anus to the posterior convergence of the fourchette in female fetuses and to the posterior base of the scrotum in male fetuses. Statistical analysis included the construction of the normal, modeled, centile, and standard deviation range, for each gestational age. Intraobserver and interobserver variability was assessed by the interclass correlation and Bland-Altman plot. Results: Adequate measurements were obtained for 218 female and 206 male fetuses. For 17 cases, measurements were not possible because of fetal lie. AGD increased linearly throughout gestational age (GA) (r2=0.808) for both sexes and was expressed by the following regression equation: for male fetuses, -12.348+1.075*GA, and for female fetuses, -3.179+0.513*GA, where GA is gestational age. The normal centiles, means, and standard deviations, per week, are presented. Conclusion: AGD measurement in utero is feasible. These measurements assess the normality of the perineal region and may assist in the detection of genital anomalies.