Author's abstract: The alligator snapping turtle (Macrochelys temminckii) is a highly aquatic turtle only found in Gulf of Mexico drainages. The biology of this species remains relatively unknown, despite years of harvest and use as a zoological attraction. The purposes of this study were to (1) determine gender size classes and assess sexual size dimorphism, (2) delineate the reproductive cycle of M. temminckii, particularly their sex steroid seasonality, and (3) evaluate possible genetic divergence between M. temminckii found in southwest Georgia and northern Florida. Alligator snapping turtles exhibit pronounced sexual size dimorphism as adults. However, determining sex in smaller individuals is difficult. The purpose of this study was to assess gender size classes and sexual dimorphism in Macrochelys temminckii. Alligator snapping turtles in southwest Georgia were trapped from March 2008 to April 2010. Captured turtles were bled, sexed based on size and ultrasound, and measured for multiple characteristics. For all measurements, adult males were larger than females. The relationships between carapace length, plastron-to-vent length, and testosterone levels were used to determine gender of smaller individuals. To ensure accurate sexual assignment when studying M. temminckii, data from multiple methods (morphological measurements, ultrasonography, testosterone levels, and, laparoscopy) should be used. To delineate the reproductive cycle of M. temminckii, blood samples were taken from captured individuals, and plasma testosterone and estradiol were measured using enzyme immunoassay. Males exhibited seasonal changes in testosterone levels, with maximum levels occurring in October. Mating behavior was elucidated from the appearance of wounds on males that only occurred in April. Females displayed seasonality in testosterone and estradiol levels, with relative peaks occurring in March and April (mating and ovulation) and September and October (follicular development). Genetic structure between M. temminckii populations separated by a dam and under different hunting pressures was assessed using microsatellite loci. Average number of alleles per locus and number of unique alleles was higher for the Florida population. Genetic structure was detected between the populations (FST = 0.043, p = 0.001). The rapid development of structuring between Florida and Georgia M. temminckii warrants further investigation into the effects of fragmentation and overexploitation on this species.