Multiple paternity in single litters (siring of offspring by >1 male) has been documented in several taxa with different mating systems. However, information on occurrence of multiple paternity in ungulates is lacking. We used 19 DNA microsatellite markers to assign parentage in 41 litters of captive white-tailed deer (Odocoileus virginianus) born in 6 pens with multiple males during 1997–1999. We detected multiple paternity in 7 of 27 litters with ≥2 offspring, occurring in 4 of 6 pens: 2 of 3 triplet litters and 5 of 24 twin litters. This is the first reported evidence of multiple paternity for single ungulate litters, which indicates that some aspects of ungulate reproductive ecology are not well understood. The occurrence of multiple paternity in litters of free-ranging ungulates would have implications for ungulate mating systems and reproductive strategies. Sex-linked microsatellite markers may provide a promising method for investigating female promiscuity in free-ranging ungulate populations where litter size is typically ≤2 offspring.