Ano-genital distance (AGD) is a sexually dimorphic trait that is a well established reproductive toxicity endpoint in animals. In male animals, a shortened AGD is associated with a variety of genital abnormalities including hypospadias and cryptorchidism. Consensus on the anatomical definition of AGD in humans remains to be established and few data exist on the determinants and normal variance in the general population. We implemented a standardized anthropometric protocol to measure AGD, ano-scrotal distance (ASD), and ano-fourchette distance (AFD) in 169 (82 male, 87 female) infants in the University of Washington newborn nursery in 2008. We collected data on the following characteristics: weight, length, and occipital head circumference, race and relevant gestational complications. Using linear regression modelling, we examined AGD/ASD/AFD for sexual dimorphism, normal population variance and predictors of the measurement in infants. The mean male and female AGD measurements were 52.0 mm (SD ± 5.5) and 37.2 mm (SD ± 3.7). The mean ASD and AFD were 23.0 mm (SD ± 3.8) and 15.1 mm (SD ± 2.9). Weight, length, occipital head circumference and gestational age were associated with AGD (p < 0.05). Weight and length were the most important correlates to AGD. We confirmed previous findings that AGD is a sexually dimorphic measurement that is most strongly predicted by infant weight. The application of this measurement to clinically relevant outcomes remains to be explored in further depth.