Purpose <p>Although adrenal vein sampling (AVS) is the standard method for subtype diagnosis in patients with primary aldosteronism (PA), the methods&#xa0;used to sample the adrenal vein are not standardized. The aim of this study was to establish the optimal method for sampling the adrenal vein based on the pathological findings after surgery for PA.</p> Methods <p>We enrolled 168 consecutive patients who were diagnosed to have PA and underwent AVS at our institution between 2019 and 2023. The impact of sampling by gentle negative pressure (GNP) on the accuracy of diagnosis of the PA subtype was compared with that of sampling by gravity, whereby blood flows out naturally.</p> Results <p>AVS was performed successfully on both sides in 139 patients using the two sampling methods. Subtype diagnosis using the two sampling methods was concordant in 128 (92.1%) of the 139 patients and discordant in 11 (7.9%). Among the 11 patients with a discordant subtype diagnosis, unilateral adrenalectomy was performed in three with the right unilateral subtype by gravity and the bilateral subtype by GNP, one with the bilateral subtype by gravity and the right unilateral subtype by GNP, and one with the left unilateral subtype by gravity and the bilateral subtype by GNP. The pathological findings after surgery showed that the false-negative rate was 20% (1/5) with data obtained by the gravity method and 80% (4/5) with data obtained by the GNP method. Bilateral AVS took significantly longer when sampling was performed by the gravity method than when it was performed by GNP (<i>p</i> &lt; 0.01).</p> Conclusions <p>The gravity method may be preferable to GNP for AVS in patients with PA.</p> Graphical Abstract <p></p>