Environmental factors are known to influence sex determination in many nonmammalian vertebrates. In all crocodilians studied thus far, temperature is the only known determinant of sex. However, the molecular mechanisms mediating the effect of temperature on sex determination are not known. Aromatase (CYP19A1) and SOX9 play critical roles in vertebrate sex determination and gonadogenesis. Here, we used a variety of techniques to investigate the potential roles of DNA methylation patterning on CYP19A1 and SOX9 expression in the American alligator, an organism that relies on temperature-dependent sex determination. Our findings reveal that developing gonads derived from embryos incubated at a male-producing temperature (MPT) show elevated CYP19A1 promoter methylation and decreased levels of gene expression relative to incubation at a female-producing temperature (FPT). The converse was observed at the SOX9 locus, with increased promoter methylation and decreased expression occurring in embryonic gonads resulting from incubation at FPT relative to that of MPT. We also examined the gonadal expression of the three primary, catalytically active DNA methyltransferase enzymes and show that they are present during critical stages of gonadal development. Together, these data strongly suggest that DNA methylation patterning is a central component in coordinating the genetic cascade responsible for sexual differentiation. In addition, these data raise the possibility that DNA methylation could act as a key mediator integrating temperature into a molecular trigger that determines sex in the alligator.