Cortisol diurnal variation may be abnormal among patients with endogenous Cushing syndrome (CS). The study objective was to compare the plasma cortisol AM/PM ratios between different etiologies of CS. This is a retrospective cohort study, conducted at a clinical research center. Adult patients with CS that underwent adrenalectomy or trans-sphenoidal surgery (n=105) were divided to those with a pathologically confirmed diagnosis of Cushing disease (n=21) and those with primary adrenal CS, including unilateral adrenal adenoma (n=28), adrenocortical hyperplasia (n=45), and primary pigmented nodular adrenocortical disease (PPNAD, n=11). Diurnal plasma cortisol measurements were obtained at 11:30 PM and midnight and at 7:30 and 8:00 AM. The ratios between the mean morning levels and mean late-night levels were calculated. Mean plasma cortisol AM/PM ratio was lower among CD patients compared to those with primary adrenal CS (1.4±0.6 vs. 2.3±1.5, p<0.001, respectively). An AM/PM cortisol ratio≥2.0 among patients with unsuppressed ACTH (>15 pg/ml) excludes CD with a 85.0% specificity and a negative predictive value (NPV) of 90.9%. Among patients with primary adrenal CS, an AM/PM cortisol≥1.2 had specificity and NPV of 100% for ruling out a diagnosis of PPNAD. Plasma cortisol AM/PM ratios are lower among patients with CD compared with primary adrenal CS, and may aid in the differential diagnosis of endogenous hypercortisolemia.