Biased sex ratios of young of birds and mammals clearly occur and may have an adaptive significance, but we rarely know the stage at which a bias is generated, or the mechanism. If a bias is generated prior to birth, studies of marsupials may be insightful because gestation is short and neonates are relatively undifferentiated. This study investigated whether biased sex ratios in Antechinus agilis are generated in the brief period between birth and the attachment of young to the mother's teats. When all young born, or just pouch young, or supernumerary young were considered, litters were strongly biased towards females (0.32 males), and there was no significant difference across the groups, so a bias is generated before birth in this species. Evidence from counts of corpora lutea suggests that embryo loss during gestation cannot account fully for the level of bias observed. Therefore, prefertilization mechanisms must contribute to the generation of sex–biased litters in this marsupial.