Heightened reactivity to ambiguous emotional stimuli, such as surprized faces, is a transdiagnostic psychopathology risk factor. Women show elevated amygdala activation to ambiguous emotional stimuli relative to men, which may underlie their significantly higher risk for mood disorders. Moreover, there are sex/gender differences in the effects of stress on both emotion processing and emotion regulation, with greater impact of stress on negative emotionality in females. We predicted that chronic stress would be associated with stronger amygdala activation to surprized faces, and these effects would be amplified in girls/women. We tested the interactions of chronic stress and gender/sex on amygdala activity in a sample of 297 healthy participants (59% girls/women, age range 6-75 years) who provided a saliva sample and who viewed emotional face stimuli while undergoing functional neuroimaging. Saliva samples were assayed for two markers of chronic stress: Epstein-Barr Virus (EBV) expression and diversity of Lactobacilli species. Among girls/women, higher chronic stress was associated with greater amygdala activation to ambiguous emotional images than lower stress exposure, although this effect was not statistically significant. Counter to predictions, the reverse effect was found among boys/men (i.e., higher stress exposure was associated with lower amygdala activation). Results were similar across left and right amygdalae, and across both stress measures. Although our findings are preliminary and should be replicated, they align with findings on gender differences in valence bias, and broadly support the hypothesis that there are gender/sex differences in the effects of chronic stress on neural reactivity to emotional ambiguity, particularly in areas of the brain sensitive to emotion regulation. Possibly, stress does not universally increase negativity, but instead amplifies default emotional biases-which for boys/men, may result in less amygdala reactivity.