Prenatal stress (PNS) can influence behaviors associated with cognition, reward and emotional regulation, which are controlled by brain areas such as the cortex, hippocampus, hypothalamus, midbrain and cerebellum. Allopregnanolone in these regions modulates behavioral and parasympathetic effects. The current study tested whether exposing pregnant dams to 5 days of resident-intruder stress on prenatal days 15-20 for 10 min altered the levels of allopregnanolone in cortex, hypothalamus, hippocampus, midbrain, and cerebellum of male and female juvenile offspring. In cortex, hypothalamus, and midbrain of male rats exposed to prenatal stress, levels of allopregnanolone were significantly lower compared to all other groups. In the hippocampus and cerebellum, among females exposed to prenatal stress levels were significantly higher compared to all other groups. These differences in allopregnanolone levels varying by prenatal stress, sex and brain regions provide insight in potential mechanism of stress regulation and etiopathophysiology of stress-related disorders. © 2020 The Authors.