The aim of the present study was to investigate the reproductive behavior of the giant Amazon River turtle (Podocnemis expansa) in the Amazon. This was carried out by estimating the degree of polymorphism in five DNA microsatellites in a sample of 359 hatchlings from 12 nests in the Mamirauá Sustainable Development Reserve in the municipality of Tefé, state of Amazonas, Brazil. An analysis of allele frequency and variation in the five microsatellite loci allowed for determination of multiple paternity in all nests sampled. Contributions of at least four male turtles to one nest, three male turtles to six nests, and two male turtles to five nests were identified. Knowledge on the reproductive behavior of P. expansa can assist in the establishment of management and conservation strategies for the population in the Mamirauá Reserve.