Background: Exposure to adversity is a risk factor for many mental and somatic health problems. Hypothalamic-pituitary-adrenal (HPA) axis dysregulation is a potential mechanism linking adversity exposure and negative health outcomes. However, associations between adversity exposure and HPA-axis activity have been inconsistent. To understand the impact of adversity on the HPA-axis, we examined associations between early-life and recent adversity with hair cortisol concentration, an indicator of long-term systemic cortisol levels. Methods: We included 1166 adult participants of the Netherlands Study of Depression and Anxiety (NESDA). Hair cortisol was measured in 3 cm of proximal hair, representing cortisol exposure during the previous 3 months. Childhood maltreatment, childhood negative life events, and recent negative life events were retrospectively assessed using interview and self-report questionnaires. Linear regression analyses were performed to assess the associations between childhood maltreatment, childhood life events and recent life events with hair cortisol. Associations with cumulative adversity exposure and with subtypes of childhood maltreatment, childhood and recent negative life events were also investigated, as were interaction effects between adversity and sex, age and psychopathology. Results: Childhood maltreatment (β = 0.034, p = 0.243), childhood life events (β = − 0.017, p = 0.544), and recent life events (β = − 0.021, p = 0.456) were not significantly associated with hair cortisol levels. Subtypes of childhood maltreatment and specific childhood and recent life events were not significantly associated with hair cortisol (pFDR>0.05). There were no significant interaction effects between adversity exposure and sex, age or depression/anxiety diagnostic status on hair cortisol. Conclusions: There were no significant associations between childhood and recent adversity with systemic cortisol levels in adults. Effects of early-life and adult adversity are complex and may not directly impact on long-term systemic cortisol levels as measured in hair.