The incidence of multiple paternities in two populations of Podocnemis expansa of Brazilian Amazon both rose in captivity and in natural habitat, was verified by using five microsatellite loci. Three hundred twenty-seven offspring from two different sampling sites were genotyped. The analysis revealed a 100% rate of multiple paternities in both populations. The Mendelian distribution of the alleles found in each nest was used to determine the number of contributing males. It was estimated that at least ten males contributed to each brood in captivity and nine contributed to each brood in the wild. These values are the highest ever recorded for the number of contributing P. expansa males. The findings have considerable implications regarding the conservation of this species, given that multiple paternities is important to the maintenance of genetic variability and has important consequences in increasing the effective size of a population in comparison to single paternities.