The aim of this study is to contribute to the determination of the normal values of human anogenital distance (AGD) and anal position index (API) in the antenatal period. 59 formalin-fixed human fetuses were examined. AGD was measured by the distance between the center of the anus and the posterior fourchette in females, and the distance between the center of the anus and the posterior scrotal raphe in males. API in female fetuses was determined with the formula API = fourchette-center of anus/fourchette-coccyx formula, and API = posterior scrotal raphe-center of anus/posterior scrotal raphe-coccyx in males. The mean AGDs of the female and male fetuses in the second trimester were 5.60 ± 1.60 mm and 9.64 ± 2.75 mm and 12.88 ± 4.14 mm and 17.26 ± 5.55 mm in the third trimester, respectively. The AGD values were found to be significantly higher in the males (p = 0.002). While the API values detected in the female and male fetuses were 0.43 ± 0.085 and 0.55 ± 0.072 in the second trimester, they were 0.46 ± 0.079 and 0.55 ± 0.058 in the third trimester. The API values were found to be significantly higher in the male fetuses (p < 0.001). When the distribution of API values of the fetuses in the second and third trimesters was examined, no significant difference was found (p = 0.499). In addition, no significant correlation was found between API and AGD values and percentile groups of fetuses (p ˃ 0.05). The AGD and API differed significantly between female and male fetuses starting from the antenatal second trimester, and the difference was preserved independently of the fetal percentile in the later stages of pregnancy.