Anogenital distance (AGD) is longer in male than in female neonatal rodents, but can be altered by the prenatal environment. The aim of this experiment was to examine the effects of the sex composition of the litter on AGD in a species with a small litter size. We found that the AGD distributions of male and female California mice, Peromyscus californicus, overlap before weaning (33 days of age), but after weaning, males have a larger AGD than females. Because AGD is significantly correlated with body mass in both males and females, we analyzed the effect of the sex composition of the litter on AGD, using ANCOVA on logarithmically transformed data, with the logarithm of body mass as the covariate. We showed that the sex composition of the litter does not affect AGD in males but has an effect on the AGD in females at birth and this effect is significant at 33 days of age. Females from litters composed of more than 75% males had a longer AGD than those born in litters composed principally of females. This study shows that intrauterine litter composition affects AGD in females of a species characterized by small litter sizes, as has been shown in rodent species with large litter sizes. Thus, AGD can be used as a predictor of masculinization of females due to intrauterine position.