OBJECTIVE: Adrenal vein sampling (AVS) is considered the gold-standard for identifying unilateral primary aldosteronism (PA), but is sensitive to any asymmetry in cortisol production. Subclinical autonomous cortisol production likely contributes to discordance between cross-sectional imaging and AVS. DESIGN AND METHODS: Retrospective chart review was performed of patients in Calgary, Alberta who (1) had a diagnosis of PA with clear adrenal mass, (2) had discordant AVS and cross-sectional imaging, and (3) underwent dexamethasone-suppressed NP59-iodocholesterol adrenal scintigraphy (n = 25). Postoperative biochemical and clinical outcomes were evaluated. Surgical pathology was analyzed with immunohistochemical staining for CYP11B1 and CYP11B2. RESULTS: NP59 scanning demonstrated autonomous steroidogenesis from the same side as the computed tomography (CT)-identified lesion, despite discordant AVS results, in 19/25 cases. Out of the 16 patients who underwent adrenalectomy (guided by NP59), 11 cases had a final diagnosis of cortisol-producing adenoma with bilateral PA, while the final diagnosis in the remaining 5 cases was cortisol-producing adenoma with unilateral PA (defined by a complete biochemical response). All cases preoperatively had low/suppressed adrenocorticotropin hormone that rose postadrenalectomy, in keeping with the resolution of cortisol autonomy. Adrenal vein sampling incorrectly diagnosed the subtype of PA or falsely localized the side of aldosterone excess in 10/16 cases. CONCLUSIONS: Discordant CT and AVS results in patients with PA and obvious adrenal mass are often explained by subclinical asymmetric cortisol excess. Clinicians should be aware of the limitations of AVS in the presence of subtle autonomous cortisol secretion and must be able to counsel patients regarding the possible outcomes from surgery when discordant lateralization is present.