Macrochelys suwanniensis is a newly described species endemic to the Suwannee River drainage in the southeastern United States. We conducted a study of M. suwanniensis in the Santa Fe River (SFR), the major Florida tributary of the Suwannee River, between 2004 and 2011. We captured 109 individuals (24% immature, 44% adult female, 32% adult male). Adult males (mean straight midline carapace length [CL] = 530.7 mm, mean mass = 34.0 kg) were significantly larger than adult females (mean CL = 424.0 mm, mean mass = 17.2 kg), with a sexual size dimorphism index of −0.25 based on mean CL. The largest turtle in our study was 623 mm CL and weighed 54.4 kg. Adult females were significantly larger (CL) in the lower SFR than in the upper SFR (these reaches are separated where the river flows underground for 5 km). All adult males > 600 mm CL were captured in the lower SFR. Adult females were proportionately heavier in the upper SFR than in the lower SFR; males did not show this difference. We hypothesize that these differences in body size are related to habitat. The M. suwanniensis population in the SFR presently appears healthy, but we suggest this species requires continued protection because of vulnerability of adults to harvest.