Genetic studies of multiple paternity are a valuable tool to gain information on the reproductive biology of turtles. We analyzed paternity type in Podocnemis sextuberculata and related number of fathers per nest to nesting period (beginning, middle, or end of nesting season); clutch size (number of eggs); female size; and hatchling success. Females were captured and maximum linear carapace lengths measured during the 60 days that encompass the nesting season at Rio Trombetas Biological Reserve (Pará, Brazil). Nests were marked and blood samples collected from hatchlings. Six heterologous loci were used: five from Podocnemis unifilis and one from Podocnemis expansa. Hatchlings were analyzed from 23 nests, and the rate of multiple paternity was 100%. The mean number of fathers per nest was six (± 0.9), and no significant difference between number of fathers in a nest and nesting period. Similarly there was no significant relationship between number of fathers in a nest and female size or hatchling success rate. Number of fathers was, however, positively correlated with clutch size (Spearman correlation rho = 0.47; P > 0.05). To our knowledge, this is the first study to test the relationship between multiple paternity and ecological aspects of the reproductive ecology of turtles in the genus Podocnemis.