In species with temperature-dependent sex determination, embryonic gonadal differentiation can be modified by exposure to exogenous chemicals such as environmental contaminants. Although phenotypic outcomes of such events are well documented, the underlying molecular mechanisms are rarely described. Here we examine the genetic and epigenetic effect of the embryonic exposure to polychlorinated biphenyls (PCBs) on gonad differentiation in red-eared slider turtles (Trachemys scripta). Some PCB congeners are without effect whereas others synergize to alter sex determination in this species. Application of two potent PCB congeners alter the physiological processes of gonad development normally dictated by the male-producing temperature (MPT), resulting sex ratios significantly biased toward female hatchlings. Of these PCB-induced females, oviduct formation is prominently distorted regardless of ovary development. Further, gonadal expression of ovarian markers, aromatase, FoxL2, and Rspo1, is activated whereas testicular markers, Dmrt1 and Sox9, are suppressed compared with typical expression patterns observed at MPT. DNA methylation profiles of the aromatase promoter in PCB-treated gonads do not follow the typical methylation pattern observed in embryos incubating at female-producing temperature. Rather, the MPT-typical methylation profiles is retained despite the induced ovarian formation. Overall, our studies demonstrate that PCB exposure alters the transcriptional profiles of genes responsible for gonadal differentiation but does not re-establish the epigenetic marks of the aromatase promoter normally set by incubation temperatures in embryonic gonads.