In general, women are at higher risk for anxiety disorders than men. A characteristic of many anxiety disorders is generalization of fear responses, which occurs when fear is expressed towards non-fearful stimuli. Previous research from our lab demonstrates that in ovariectomized female rats, estradiol induces contextual fear generalization, whereas in gonadectomized males, estradiol reduces generalization. Our lab determined that estradiol acts within the dorsal CA1 (dCA1) and anterior cingulate cortex (ACC) to induce generalization in females. However, in males the brain regions mediating estradiol’s ability to reduce generalization are unknown. Given the influence estradiol has on glutamate receptors, we hypothesized that estradiol induces generalization in female rats by enhancing glutamatergic signaling in the dCA1 and ACC. Using a passive avoidance paradigm, we examined the role of NMDA and AMPA type glutamate receptors in the dCA1 and ACC and found that both are necessary for estradiol-induced generalization. Pharmacological blockade of both receptor types reduced generalization caused by administration of estradiol. To determine specific brain regions involved in the reduction of generalization in male rats, we examined the effect of estradiol in the dCA1 and the bed nucleus stria terminals (BNST), two regions important for mediating the behavioral effects of estradiol. Results demonstrated estradiol infusions into the dCA1, but not the BNST, reduced generalization in males. These data suggest a similar neural circuit is involved in the sex-specific effects of estradiol on generalized fear. Understanding the sex-dependent influences of estradiol on generalization will allow for improved, sex-specific, treatments for many anxiety disorders.