Killifish (Fundulus heteroclitus) inhabiting the New Bedford Harbor (NBH) Superfund Site have evolved resistance to the toxic and biochemical effects of non-ortho (dioxin-like) polychlorinated biphenyls (DL-PCBs) and other compounds that act via the aryl hydrocarbon receptor (AHR) signaling pathway. However, the majority of PCBs in NBH are ortho-substituted (non-DL) PCBs (o-PCBs), and the impacts of these o-PCBs on fish populations are not well understood. To determine whether the NBH killifish population has adapted to o-PCBs, we performed a series of experiments involving exposure to killifish embryos and adults from NBH and a reference site (Scorton Creek SC) to 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB-153), a model o-PCB. PCB-153 was not acutely embryotoxic to developing F2 killifish embryos (SC or NBH) at concentrations up to 28 µM. RNA-seq showed that SC embryos exposed to PCB-153 (28 µM for 6 hr at 10 days post fertilization) had changes in the expression of genes involved in glucose homeostasis. However, NBH embryos were much less sensitive to these effects of PCB-153. When adult killifish from SC and NBH were exposed to PCB-153 (20 mg/kg) and sampled 3 days later for gene expression, many more genes were affected in forebrains of SC fish than in NBH fish, in a sex-specific manner. Together, these results demonstrate that NBH killifish have evolved reduced sensitivity to o-PCBs, suggesting complex adaptation to chemical mixtures at a Superfund site. Overall design: We sequenced 5 pools of killifish embryos (biological replicates) exposed to DMSO and PCB153.