The success rate of cannulation of the right adrenal vein is limited. The aldosterone gradient within the same adrenal vein branch is specific for aldosterone-producing adenoma. This study was performed to investigate whether the absolute aldosterone gradient within the left adrenal vein (left-AV absolute aldosterone gradient) indicates unilateral excess aldosterone. A retrospective cross-sectional study in a single referral centre. In total, 123 consecutive patients with primary aldosteronism who had successful adrenal vein sampling (AVS) data were examined. The left-AV absolute aldosterone gradient was considered significant when a gradient of >4:1 in the aldosterone-to-cortisol ratio between the common trunk vein and central vein was found. The prevalence of the unilateral subtype in patients with a significant left-AV absolute aldosterone gradient. The prevalence of the unilateral subtype was higher in patients with than without a significant left-AV absolute aldosterone gradient (88.2% [15/17] vs 21.7% [23/106], P<.001). Of 60 patients with spontaneous hypokalemia, left unilateral disease on computed tomography, or both, a significant left-AV absolute aldosterone gradient was present only in patients with the unilateral subtype on AVS (42.9% [15/35]), but not in those with the bilateral subtype (0.0% [0/25]). These data were validated in an external cohort. The presence of a significant left-AV absolute aldosterone gradient can be used to diagnose the left unilateral subtype of primary aldosteronism on AVS in patients with spontaneous hypokalemia, left unilateral disease on computed tomography or both.