The giant South American river turtle (Podocnemis expansa) is the largest extant member of the suborder Pleurodira and the largest freshwater chelonian in South America. Owing to its size, its meat is sought for consumption and trade in the Amazon region. The aim of the present study was to investigate the reproductive behavior of 2 different populations of P. expansa. Allelic frequency variation was studied in 6 DNA microsatellite loci of recently hatched offspring from 8 nests on a beach in the municipality of Oriximiná in the state of Pará, Brazil. Multiple paternity was identified in all nests studied. Between 5 and 10 fathers contributed to each nest. Compared with previous studies, a greater frequency of multiple paternity and a greater number of fathers contributing to each nest were found in the present investigation. The results suggest that populations from different locations may exhibit different paternity patterns due to ecological or biological differences. By comparison with previous studies, we suggest that the differences in the number of nests, proportion of offspring per nest, and number of loci analyzed in each study may influence the frequency of multiple paternity detected.