Context: Intrinsic imperfections and lack of reliable biomarkers preclude optimal individual dosing of hydrocortisone replacement in adrenal insufficiency (AI). However, the clinical relevance of optimal dosing is exemplified by frequently occurring side effects of overreplacement and the dangers of underreplacement. Cortisol in scalp hair has been identified as a retrospective biomarker for long-term cortisol exposure. We compared hair cortisol concentrations (CORThair) of patients with primary or secondary AI on replacement therapy with those of patient controls with a pituitary disease without AI (PCs) and of healthy controls (HCs). Methods: In this cross-sectional study, hair samples and anthropometric data were collected in 132 AI patients (52 males), 42 PCs (11 males), and 195 HCs (90 males). The proximal 3 cm of hair were used. CORThair were measured using an ELISA. Results: CORThair were higher in AI patients than in HCs and PCs (P <.001), and hydrocortisone dose correlated with CORThair (P =.04). Male AI patients demonstrated higher CORThair than female patients (P <.001). AI patients had higher body mass index (BMI) than HCs (P <.001), and BMI correlated with CORThair in the whole sample (P <.001). Conclusion: Physiological hydrocortisone replacement is associated with increased CORThair. The association between CORThair and BMI could suggest a mild overtreatment that may lead to adverse anthropomorphic side effects, especially in males. CORThair measurements may be a promising additional tool to monitor cumulative hydrocortisone replacement in AI.