Polyandry has been demonstrated in a number of shark species. Studies based on a small number of loci typically estimate the minimum number of sires, which may be much lower than the actual number of sires due to the sharing of alleles by parents. We genotyped three litters of Nurse Shark (Ginglymostoma cirratum) ranging in size from 29 to 39 pups at 12 DNA microsatellite loci to determine the number of sires for each litter. We analyzed the genotypes using two analytical methods, both of which indicated that each litter had five to seven sires with one to 17 pups per sire. Previous analyses of these litters based on MHC loci indicated a minimum of four sires. Because G. cirratum are not capable of long-term sperm storage and ovulate over several weeks, they may require multiple inseminations to fertilize an entire litter. The varying levels of developmental stage among embryos found within a single litter may reflect multiple fertilizations over a prolonged period. Exact enumeration of sires as demonstrated here can provide valuable information for understanding reproductive ecology of live-bearing vertebrates.