Dichlorodiphenyltrichloroethane (DDT) is used for malaria control by 10 countries, nine of which are in Africa. Technical DDT contains various isomers with 65-80% insecticidal p,p'-DDT and 15-21% o,p'-DDT, an estrogenic chemical, while the persistent metabolite of p,p'-DDT, dichlorodiphenyldichloroethylene (p,p'-DDE), is an antiandrogen. In utero antiandrogenic exposure reduces anogenital distance in animal models and the anal position index in a single study. This study examined the associations between mother's serum DDT and DDE levels at delivery and anogenital distance in their children at birth and age 1 year. Data were collected as part of the Venda Health Examination of Mothers, Babies and their Environment (VHEMBE), a birth cohort study located in rural South Africa. DDT and DDE concentrations were measured in blood samples collected from 752 mothers at delivery. Anogenital distance measurements, taken at birth (n = 671) and age 1 year (n = 674), included anofourchette and anoclitoral distances in girls, and anoscrotal and anopenile lengths in boys. We also measured anococcygeal and coccyx-fourchette distances in girls, while in boys, we measured anococcygeal and coccyx-scrotal distances as well as penile length and penile width. The anal position index is calculated for both sexes as anoscrotal/coccyx-scrotal in boys and anofourchette/coccyx-fourchette in girls. We found no associations between p,p'-DDT/-DDE or o,p'-DDT and anogenital distance measurements at birth in either boys or girls. At 1 year, o,p'-DDE was negatively associated with anofourchette in girls (β =-1.32 mm, 95% confidence interval (CI) = -2.27, -0.38) and positively associated with penile width in boys (β = 0.30 mm, 95% CI = 0.00, 0.60). The results do not suggest an overt antiandrogenic or estrogenic effect on anogenital distance after long-term DDT exposure. These weak associations may be due to chance.