Objective and design: Adrenal venous sampling (AVS) is critical to determine the subtype of primary aldosteronism (PA). Central AVS (C-AVS) -that is, the collection of effluents from bilateral adrenal central veins (CV) -sometimes does not allow differentiation between bilateral aldosterone-producing adenomas (APA) and idiopathic hyperaldosteronism. To establish the best treatment course, we have developed segmental AVS (S-AVS); that is, we collect effluents from the tributaries of CV to determine the intra-adrenal sources of aldosterone overproduction. We then evaluated the clinical utility of this novel approach in the diagnosis and treatment of PA. Methods: We performed C-AVS and/or S-AVS in 297 PA patients and assessed the accuracy of diagnosis based on the results of C-AVS (nZ138, 46.5%) and S-AVS (nZ159, 53.5%) by comparison with those of clinicopathological evaluation of resected specimens.Results: S-AVS demonstrated both elevated and attenuated secretion of aldosterone from APA and non-tumorous segments, respectively, in patients with bilateral APA and recurrent APA. These findings were completely confirmed by detailed histopathological examination after surgery. S-AVS, but not C-AVS, also served to identify APA located distal from the CV. Conclusions: Compared to C-AVS, S-AVS served to identify APA in some patients, and its use should expand the pool of patients eligible for adrenal sparing surgery through the identification of unaffected segments, despite the fact that S-AVS requires more expertise and time. Especially, this new technique could enormously benefit patients with bilateral or recurrent APA because of the preservation of non-tumorous glandular tissue.