Geological processes influence water chemistry and biological productivity along the Suwannee River and divide the mainstem of the river into 6 distinct ecological reaches (ERs) in Florida. Because of these distinctions, we hypothesized that the Macrochelys suwanniensis (Suwannee Alligator Snapping Turtle) population varies among ERs. During 2011–2013, we sampled two 5-km sites in each ER to investigate spatial variation in relative abundance, sex ratio, size-class distribution, and body size of M. suwanniensis. Capture rates and male body sizes were greater in the middle ERs than in the upstream and downstream ERs. Sex ratios were male-skewed only in the middle ERs. Additional research is needed to understand the ecological drivers of M. suwanniensis population dynamics in this river.