In the blue crab Callinectes sapidus (Rathbun, 1896), double ejaculations have been observed; however, it is not known if these multiple inseminations are the products of different males or if they lead to effective multiple paternities. In this study, 75 mature females and their spermathecae were genotyped for five polymorphic microsatellite loci. The incidence of multiple paternity was analyzed by comparing the genotypes of either spermathecae or individual ejaculates with the corresponding mother, and the male contribution was then deduced. Females contained double ejaculates (5.3%),which genotyping confirmed as the products of different males. In addition, a routine screening of 68 crabs from a single hatchery brood at two loci indicated more than two parents. Mitochondrial analysis confirmed that the brood shares the same mother; therefore, the extra alleles must be due to different fathers. Our findings indicate that female blue crabs are not only capable of mating with more than one male but that multiple inseminations can lead to multiple paternity within a brood.